Compstatin Analogs with Improved Pharmacokinetic Properties

ABSTRACT

Compounds comprising peptides capable of binding C3 protein and inhibiting complement activation are disclosed. The compounds comprise compstatin analogs in which the N-terminus contains an added or substituted component that improves (1) the peptide&#39;s binding affinity to C3 or its fragments, (2) the peptide&#39;s solubility in aqueous liquids, (3) the peptide&#39;s plasma stability, (4) the peptide&#39;s in vivo retention and/or (5) the peptide&#39;s bioavailability, as compared with an unmodified compstatin peptide under equivalent conditions. Pharmaceutical compositions and methods of using the compounds are also disclosed.

GOVERNMENT SUPPORT

Pursuant to 35 U.S.C. §202(c), it is acknowledged that the United Statesgovernment may have certain rights in the invention described herein,which was made in part with funds from the National Institutes of Healthunder Grant Nos. GM 62134, AI30040, AI068730, GM097747 and EY020633.

FIELD OF THE INVENTION

This invention relates to activation of the complement cascade in thebody. In particular, this invention provides peptides andpeptidomimetics that bind the C3 protein with nanomolar affinity andinhibit complement activation, exhibit robust aqueous solubility, plasmastability and in vivo retention and are bioavailable by multiple routesof administration.

BACKGROUND OF THE INVENTION

Various publications, including patents, published applications,technical articles and scholarly articles are cited throughout thespecification. Each of these cited publications is incorporated byreference herein, in its entirety.

The human complement system is a powerful player in the defense againstpathogenic organisms and the mediation of immune responses. Complementcan be activated through three different pathways: the classical,lectin, and alternative pathways. The major activation event that isshared by all three pathways is the proteolytic cleavage of the centralprotein of the complement system, C3, into its activation products C3aand C3b by C3 convertases. Generation of these fragments leads to theopsonization of pathogenic cells by C3b and iC3b, a process that rendersthem susceptible to phagocytosis or clearance, and to the activation ofimmune cells through an interaction with complement receptors(Markiewski & Lambris, 2007, Am J Pathol 171: 715-727). Deposition ofC3b on target cells also induces the formation of new convertasecomplexes and thereby initiates a self-amplification loop.

An ensemble of plasma and cell surface-bound proteins carefullyregulates complement activation to prevent host cells from self-attackby the complement cascade. However, excessive activation orinappropriate regulation of complement can lead to a number ofpathologic conditions, ranging from autoimmune to inflammatory diseases(Holers, 2003, Clin Immunol 107: 140-51; Markiewski & Lambris, 2007,supra; Ricklin & Lambris, 2007, Nat Biotechnol 25: 1265-75; Sahu et al.,2000, J Immunol 165: 2491-9). The development of therapeutic complementinhibitors is therefore highly desirable. In this context, C3 and C3bhave emerged as promising targets because their central role in thecascade allows for the simultaneous inhibition of the initiation,amplification, and downstream activation of complement (Ricklin &Lambris, 2007, supra).

Compstatin was the first non-host-derived complement inhibitor that wasshown to be capable of blocking all three activation pathways (Sahu etal., 1996, J Immunol 157: 884-91; U.S. Pat. No. 6,319,897). This cyclictridecapeptide binds to both C3 and C3b and prevents the cleavage ofnative C3 by the C3 convertases. Its high inhibitory efficacy wasconfirmed by a series of studies using experimental models that pointedto its potential as a therapeutic agent (Fiane et al., 1999a,Xenotransplantation 6: 52-65; Fiane et al., 1999b, Transplant Proc31:934-935; Nilsson et al., 1998 Blood 92: 1661-1667; Ricklin & Lambris,2008, Adv Exp Med Biol 632: 273-292; Schmidt et al., 2003, J BiomedMater Res A 66: 491-499; Soulika et al., 2000, Clin Immunol 96:212-221). Progressive optimization of compstatin has yielded analogswith improved activity (Ricklin & Lambris, 2008, supra; WO2004/026328;WO2007/062249). One of these analogs is currently being tested inclinical trials for the treatment of age-related macular degeneration(AMD), the leading cause of blindness in elderly patients inindustrialized nations (Coleman et al., 2008, Lancet 372: 1835-1845;Ricklin & Lambris, 2008, supra). In view of its therapeutic potential inAMD and other diseases, further optimization of compstatin to achieve aneven greater efficacy is of considerable importance.

Earlier structure-activity studies have identified the cyclic nature ofthe compstatin peptide and the presence of both β-turn and hydrophobiccluster as key features of the molecule (Morikis et al., 1998, ProteinSci 7: 619-627; WO99/13899; Morikis et al., 2002, J Biol Chem277:14942-14953; Ricklin & Lambris, 2008, supra). Hydrophobic residuesat positions 4 and 7 were found to be of particular importance, andtheir modification with unnatural amino acids generated an analog with264-fold improved activity over the original compstatin peptide(Katragadda et al., 2006, J Med Chem 49: 4616-4622; WO2007/062249).

While previous optimization steps have been based on combinatorialscreening studies, solution structures, and computational models (Chiuet al., 2008, Chem Biol Drug Des 72: 249-256; Mulakala et al., 2007,Bioorg Med Chem 15: 1638-1644; Ricklin & Lambris, 2008, supra), thepublication of a co-crystal structure of compstatin complexed with thecomplement fragment C3c (Janssen et al., 2007, J Biol Chem 282:29241-29247; WO2008/153963) represents an important milestone forinitiating rational optimization. The crystal structure revealed ashallow binding site at the interface of macroglobulin (MG) domains 4and 5 of C3c and showed that 9 of the 13 amino acids were directlyinvolved in the binding, either through hydrogen bonds or hydrophobiceffects. As compared to the structure of the compstatin peptide insolution (Morikis et al., 1998, supra), the bound form of compstatinexperienced a conformational change, with a shift in the location of theβ-turn from residues 5-8 to 8-11 (Janssen et al., 2007, supra;WO2008/153963).

The present inventors recently developed a series of compstatin analogswith improved potency based on N-methylation of the peptide backbone,particularly at position 8 of the peptide, and substitutions at theflanking position 13 (Qu et al., 2011, Molec Immunol 48: 481-489,WO2010/127336). Those modifications were reported to produce acompstatin analog with improved binding affinity over the most activeanalogs reported to date.

Compstatin and its analogs have significant potential for clinicalapplications. Recent examples include the reduction of filter-inducedadverse effects during hemodialysis and organ preservation in sepsis.Importantly, the intravitreal use of compstatin analogs has shownpromising results in the treatment of age-related macular degeneration(AMD), both in non-human primate (NHP) studies and in phase I clinicaltrials. The low molecular weight of compstatin and its analogs, theirhigh specificity and efficacy, and their ability to simultaneouslyinhibit all complement activation and amplification pathways contributeto a beneficial drug profile. Extended clinical applications (e.g.,systemic administration by a variety of routes), however, placeadditional demands on the molecular properties of compstatinderivatives. For instance, disfavored pharmacokinetic profiles due torapid elimination from plasma still impose a major limitation for theclinical use of peptidic drugs. Additionally, though oral delivery isthe most convenient and popular route of drug administration, mostpeptide drugs display little or no oral activity. This is believed to bedue mainly to degradation in the gastrointestinal tract by enzymes andextreme conditions, as well as poor permeability of the intestinalmucosa. Consequently, most protein-based therapeutics are administeredby frequent injections through the parenteral routes such as byintravenous, intramuscular and subcutaneous injection. These forms ofadministration are costly and can require a medical professional, all ofwhich can result in poor patient acceptance and compliance. In view ofthe foregoing, it is clear that the development of modified compstatinpeptides or mimetics with greater activity, in vivo stability, plasmaresidence time and bioavailability would constitute a significantadvance in the art.

SUMMARY OF THE INVENTION

The present invention provides analogs of the complement-inhibitingpeptide, compstatin, which maintain improved complement-inhibitingactivity as compared to compstatin, and which also possess improvedsolubility and stability and pharmacokinetic properties, includingbioavailability via multiple routes of administration.

One aspect of the invention features a compound comprising a modifiedcompstatin peptide (ICVVQDWGHHRCT (cyclic C2-C12; SEQ ID NO:1) or analogthereof, wherein the modification comprises an added or substitutedN-terminal component that improves (1) the peptide's C3, C3b or C3cbinding affinity, (2) the peptide's solubility in aqueous liquids,and/or (3) the peptide's plasma stability and/or plasma residence time,as compared with an unmodified compstatin peptide under equivalentconditions.

Components that can be added to the N-terminus of the peptide compriseamino acid residues other than L-Gly, or peptidic or non-peptidicanalogs of such amino acids. In certain embodiments, the added componentis a D-amino acid, and/or the component can include at least onearomatic ring. In one embodiment, the added component is D-Tyr. In otherembodiments, the added component comprises an N-methylated amino acid.In one embodiment, the N-methylated amino acid is N-methylated L-Gly,also referred to herein as Sar. Thus, in various embodiments, the addedcomponent is D-Tyr, D-Phe, Tyr(Me), D-Trp, Tyr, D-Cha, Cha, Phe, Sat,Arg, mPhe, mVal, Trp, mIle, D-Ala, mAla, Thr or Tyr.

In other embodiments, the modified compstatin peptide comprises asubstituted N-terminal component wherein Ile at position 1 is replacedwith Ac-Trp or a dipeptide Tyr-Gly.

The compound can also include other modifications. For instance, His atposition 9 (based on numbering of compstatin) can be replaced with Ala.Additionally, Val at position 4 can be replaced with Trp or an analog ofTrp. Particular analogs of Trp at position 4 include 1-methyl Trp or1-formyl Trp. The Trp at position 7 can also be replaced with an analogof Trp, including but not limited to a halogenated Trp. Othermodifications include modification of Gly at position 8 to constrain thebackbone conformation at that location. In particular, the backbone canbe constrained by replacing the Gly at position 8 (Gly8) withN^(α)-methyl Gly (Sar). Other modifications include replacing the Thr atposition 13 with Ile, Leu, Nle, N-methyl Thr or N-methyl Ile. Stillother modifications include replacing the disulfide bond between C2 andC12 with a thioether bond to form a cystathionine or a lantithionine.Yet another modification includes replacing the Arg at position 11 withOrn, and/or replacing the Asp at position 6 with Asn.

In particular embodiments, the compstatin analog comprises a peptidehaving a sequence of SEQ ID NO:29, which is: Xaa1-Xaa2-Cys-Val-Xaa3Gln-Xaa4 Xaa5-Gly-Xaa6-His-Xaa7-Cys-Xaa8, in which Gly between Xaa5 andXaa6 (position 8 of compstatin) optionally is modified to constrain thebackbone conformation, and wherein: Xaa1 is absent or is Tyr, D-Tyr orSar; Xaa2 is Ile, Gly or Ac-Tip; Xaa3 is Trp or an analog of Trp,wherein the analog of Trp has increased hydrophobic character ascompared with Trp; Xaa4 is Asp or Asn; Xaa5 is Trp or an analog of Tipcomprising a chemical modification to its indole ring wherein thechemical modification increases the hydrogen bond potential of theindole ring; Xaa6 is His, Ala, Phe or Trp; Xaa7 is Arg or Orn; and Xaa8is Thr, Ile, Leu, Nle, N-methyl Thr or N-methyl Ile, wherein a carboxyterminal —OH of any of the Thr, Ile, Leu, Nle, N-methyl Thr or N-methylIle optionally is replaced by —NH₂, and wherein the peptide is cyclicvia a Cys-Cys or thioether bond.

Particular embodiments of the analog include the following features: theGly at position 8 is N-methylated; Xaa1 is D-Tyr or Sar; Xaa2 is Ile;Xaa3 is Trp, 1-methyl-Trp or 1-formyl-Trp; Xaa5 is Trp; Xaa6 is Ala; andXaa8 is Thr, Ile, Leu, Nle, N-methyl Thr or N-methyl Ile with optionalreplacement of the carboxy terminal —OH with —NH₂. More specifically,Xaa8 can be Ile, N-methyl Thr or N-methyl Ile with optional replacementof the carboxy terminal —OH with —NH₂. Exemplary analogs include SEQ IDNO:7 and SEQ ID NO:18. Another aspect of the invention features acompound that inhibits complement activation, comprising a non-peptideor partial peptide mimetic of SEQ ID NO:7 or SEQ ID NO:18, wherein thecompound binds C3 and inhibits complement activation with at least500-fold greater activity than does a peptide comprising SEQ ID NO:1under equivalent assay conditions.

Another aspect of the invention features a compound as described above,which includes an additional component that extends the in vivoretention (i.e., residence time) of the compound. In one embodiment, theadditional component is polyethylene glycol (PEG). In other embodiments,the additional component is an albumin binding small molecule or analbumin binding peptide. In particular embodiments, the albumin bindingsmall molecule or albumin binding peptide is attached to the peptide atthe N- or C-terminus. The attachment can be direct or through a linkeror spacer.

Another aspect of the invention features a pharmaceutical compositioncomprising any of the above-described compounds and a pharmaceuticallyacceptable carrier. In one embodiment, the pharmaceutical composition isformulated for oral administration. In another embodiment, it isformulated for topical administration. In another embodiment, it isformulated for pulmonary administration. In another embodiment, thepharmaceutical composition is formulated for subcutaneous orintramuscular injection. In another embodiment, it is formulated forintravenous injection or infusion.

Another aspect of the invention provides for the use of any of theabove-described compounds for inhibition of complement activation invivo, ex vivo, in situ or in vitro, as well as for use in themanufacture of a medicament for the inhibition of complement activation.

Various features and advantages of the present invention will beunderstood by reference to the detailed description, drawings andexamples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Interaction of compstatin analogs with C3b. (A) Kinetic profilesof compstatin lead compounds 4 (1 MeW) (lowest, narrowest set of peaks),Cp20 (SEQ ID NO:3) (intermediate set of peaks), and peptide 14 (Cp40(SEQ ID NO:18)) (highest, broadest set of peaks) as determined by singlecycle kinetic analysis using surface plasmon resonance. (B) Rate plot ofpeptides 1-20 as well as reference compounds 4 (1 MeW) and Cp20 (SEQ IDNO:3) with isoaffinity lines shown as dashed lines. Benchmark lines forthe rate constant and affinity of Cp20 (SEQ ID NO:3) are shown.

FIG. 2. Correlation between free energy values (ΔG) as calculated from acomputational docking experiment between compstatin analogs and C3c (yaxis) and from the experimentally determined affinity values of the sameanalogs for C3b (x axis). Peptide numbers are shown next to each mark onthe plot. The correlation over the entire data set is shown as a solidline whereas the dotted line represents the correlation after exclusionof peptides 1, 5 and 7.

FIG. 3. Docking of compstatin analogs into the binding site of C3c. (A)Docked conformation of peptide 14 (Cp40 (SEQ ID NO:18), cyan in thecolor figure) and peptide 4 (gray in the color figure) (note in thenon-color figure, the aromatic ring of the dY side chain (CP40 (SEQ IDNO:18)) is superimposed in front of the ring of Y (peptide 4) and can bedistinguished in that manner). Other D-amino acids have a similarconformation as peptide 14 in the docked models. Side chains of otherresidues in peptide 4 were omitted for clarity. (B) Docking conformationof peptide 19.

FIG. 4. Stability of peptide 3 (Cp30 (SEQ ID NO:7)) and peptide 14 (Cp40(SEQ ID NO:18)) in human plasma at 37° C. Cp30 (SEQ ID NO:7), Cp40 (SEQID NO:18) and a positive control peptide 2B were spiked in human plasmato a reach a final concentration of 20 μM. The plasma was incubated at37° C. and 100 μL of sample was taken at various time points. Peptideswere extracted from plasma using solid phase extraction and analyzedusing UPLC-MS. 3A: the area of each sample at different time point wasplotted over time (square: Cp40 (SEQ ID NO:18), circle: Cp30 (SEQ IDNO:7), triangle: control peptide 2B). 3B: Chromatograph of samples fromtime 0 (Top), 24 h (middle) and 120 h (bottom).

FIG. 5. Pharmacokinetic assessment of compstatin analogs in non-humanprimates. (A) Linear plot of peptide level over time after i.v. bolusinjection of 2 mg/kg in cynomolgus monkeys, showing a biphasic modelwith a rapid initial elimination phase followed by a slow log-linearterminal phase. Cp20 (SEQ ID NO:3)—lower two lines; Cp30 (SEQ ID NO:7)(Peptide 3)—middle two lines; Cp40 (SEQ ID NO:18) (Peptide 14)—upper twolines. (B) Calculation of the plasma elimination half-life (t_(1/2))from the terminal phase (1-24 h). Cp20 (SEQ ID NO:3)—lower two lines;Cp30 (SEQ ID NO:7) (Peptide 3)—middle two lines; Cp40 (SEQ ID NO:18)(Peptide 14)—upper two lines. Dashed lines mark the range of measuredplasma levels of the target protein C3 in both panels A and B. (C)Superimposition of kinetic binding profiles of analog Cp20 (SEQ ID NO:3)to immobilized C3 from humans, baboons, cynomolgus monkeys and rhesusmonkeys as assessed by SPR.

FIG. 6. Plasma concentrations of compstatin analogCp40 (SEQ ID NO:18)following a single administration of the analog by two different routesin cynomolgus monkeys. Plasma concentrations were measured by massspectrometry at time points after subcutaneous injection (top panel) ororal administration (bottom panel).

FIG. 7. Plasma concentrations and complement inhibitory activity ofcompstatin analogCp40 (SEQ ID NO:18) following a single administrationof the analog by intramuscular injection in a baboon. Plasmaconcentrations were measured by mass spectrometry at time points afterintramuscular injection (circles). Inhibition of complement activationvia the alternative pathway was measured by an erythrocyte hemolyticassay (triangles).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Definitions

Various terms relating to the methods and other aspects of the presentinvention are used throughout the specification and claims. Such termsare to be given their ordinary meaning in the art unless otherwiseindicated. Other specifically defined terms are to be construed in amanner consistent with the definition provided herein.

The following abbreviations may be used herein: Ac, acetyl group; DCM,dichloromethane; DIC, 1,3-diisopropylcarbodiimide; DIPEA,N,N-diisopropylethylamine; DPBS, Dulbecco's Phosphate Buffered Saline;ELISA, enzyme-linked immunosorbent assay; ESI, electrospray ionization;Fmoc, 9-fluorenylmethoxycarbonyl; HOAt, 1-hydroxy-7-aza-benzotriazole;ITC, Isothermal titration calorimetry; MALDI, matrix-assisted laserdesorption ionization; MBHA, 4-methylbenzhydrylamine; NMP,N-methylpyrrolidinone; Sar, N-methyl glycine; SPR, surface plasmonresonance; TIPS, triisopropylsilane; Trt, trityl; WFI, water forinjection.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or ±10%, in some embodiments ±5%, in someembodiments ±1%, and in some embodiments ±0.1% from the specified value,as such variations are appropriate to make and used the disclosedcompounds and compositions.

The term “compstatin” as used herein refers to a peptide comprising SEQID NO:1, ICVVQDWGHHRCT (cyclic C2-C12 by way of a disulfide bond). Theterm “compstatin analog” refers to a modified compstatin comprisingsubstitutions of natural and/or unnatural amino acids, or amino acidanalogs, as well as modifications within or between various amino acids,as described in greater detail herein, and as known in the art. Whenreferring to the location of particular amino acids or analogs withincompstatin or compstatin analogs, those locations are sometimes referredto as “positions” within the peptide, with the positions numbered from 1(Ile in compstatin) to 13 (Thr in compstatin). For example, the Glyresidue occupies “position 8.”

The terms “pharmaceutically active” and “biologically active” refer tothe ability of the compounds of the invention to bind C3 or fragmentsthereof and inhibit complement activation. This biological activity maybe measured by one or more of several art-recognized assays, asdescribed in greater detail herein.

As used herein, “alkyl” refers to an optionally substituted saturatedstraight, branched, or cyclic hydrocarbon having from about 1 to about10 carbon atoms (and all combinations and subcombinations of ranges andspecific numbers of carbon atoms therein), with from about 1 to about 7carbon atoms being preferred. Alkyl groups include, but are not limitedto, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl,n-pentyl, cyclopentyl, isopentyl, neopentyl, n-hexyl, isohexyl,cyclohexyl, cyclooctyl, adamantyl, 3-methylpentyl, 2,2-dimethylbutyl,and 2,3-dimethylbutyl. The term “lower alkyl” refers to an optionallysubstituted saturated straight, branched, or cyclic hydrocarbon havingfrom about 1 to about 5 carbon atoms (and all combinations andsubcombinations of ranges and specific numbers of carbon atoms therein).Lower alkyl groups include, but are not limited to, methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, cyclopentyl,isopentyl and neopentyl.

As used herein, “halo” refers to F, Cl, Br or I.

As used herein, “alkanoyl”, which may be used interchangeably with“acyl”, refers to an optionally substituted straight or branchedaliphatic acylic residue having from about 1 to about 10 carbon atoms(and all combinations and subcombinations of ranges and specific numbersof carbon atoms therein), with from about 1 to about 7 carbon atomsbeing preferred Alkanoyl groups include, but are not limited to, formyl,acetyl, propionyl, butyryl, isobutyryl, pentanoyl, isopentanoyl,2-methyl-butyryl, 2,2-dimethylpropionyl, hexanoyl, heptanoyl, octanoyl,and the like. The term “lower alkanoyl” refers to an optionallysubstituted straight or branched aliphatic acylic residue having fromabout 1 to about 5 carbon atoms (and all combinations andsubcombinations of ranges and specific numbers of carbon atoms therein.Lower alkanoyl groups include, but are not limited to, formyl, acetyl,n-propionyl, iso-propionyl, butyryl, isobutyryl, pentanoyl,iso-pentanoyl, and the like.

As used herein, “aryl” refers to an optionally substituted, mono- orbicyclic aromatic ring system having from about 5 to about 14 carbonatoms (and all combinations and subcombinations of ranges and specificnumbers of carbon atoms therein), with from about 6 to about 10 carbonsbeing preferred. Non-limiting examples include, for example, phenyl andnaphthyl.

As used herein, “aralkyl” refers to alkyl as defined above, bearing anaryl substituent and having from about 6 to about 20 carbon atoms (andall combinations and subcombinations of ranges and specific numbers ofcarbon atoms therein), with from about 6 to about 12 carbon atoms beingpreferred. Aralkyl groups can be optionally substituted. Non-limitingexamples include, for example, benzyl, naphthylmethyl, diphenylmethyl,triphenylmethyl, phenylethyl, and diphenylethyl.

As used herein, the terms “alkoxy” and “alkoxyl” refer to an optionallysubstituted alkyl-O-group wherein alkyl is as previously defined.Exemplary alkoxy and alkoxyl groups include methoxy, ethoxy, n-propoxy,i-propoxy, n-butoxy, and heptoxy, among others.

As used herein, “carboxy” refers to a —C(═O)OH group.

As used herein, “alkoxycarbonyl” refers to a —C(═O)O-alkyl group, wherealkyl is as previously defined.

As used herein, “aroyl” refers to a —C(═O)-aryl group, wherein aryl isas previously defined. Exemplary aroyl groups include benzoyl andnaphthoyl.

Typically, substituted chemical moieties include one or moresubstituents that replace hydrogen at selected locations on a molecule.Exemplary substituents include, for example, halo, alkyl, cycloalkyl,aralkyl, aryl, sulfhydryl, hydroxyl (—OH), alkoxyl, cyano (—CN),carboxyl (—COOH), acyl (alkanoyl: —C(═O)R); —C(═O)O-alkyl, aminocarbonyl(—C(═O)NH₂), —N— substituted aminocarbonyl (—C(═O)NHR″), CF₃, CF₂CF₃,and the like. In relation to the aforementioned substituents, eachmoiety R″ can be, independently, any of H, alkyl, cycloalkyl, aryl, oraralkyl, for example.

As used herein, “L-amino acid” refers to any of the naturally occurringlevorotatory alpha-amino acids normally present in proteins or the alkylesters of those alpha-amino acids. The term D-amino acid” refers todextrorotatory alpha-amino acids. Unless specified otherwise, all aminoacids referred to herein are L-amino acids.

“Hydrophobic” or “nonpolar” are used synonymously herein, and refer toany inter- or intra-molecular interaction not characterized by a dipole.

“PEGylation” refers to the reaction in which at least one polyethyleneglycol (PEG) moiety, regardless of size, is chemically attached to aprotein or peptide to form a PEG-peptide conjugate. “PEGylated meansthat at least one PEG moiety, regardless of size, is chemically attachedto a peptide or protein. The term PEG is generally accompanied by anumeric suffix that indicates the approximate average molecular weightof the PEG polymers; for example, PEG-8,000 refers to polyethyleneglycol having an average molecular weight of about 8,000 Daltons (org/mol).

As used herein, “pharmaceutically acceptable salts” refers toderivatives of the disclosed compounds wherein the parent compound ismodified by making acid or base salts thereof. Examples ofpharmaceutically-acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. Thus, the term “acid addition salt” refers to the correspondingsalt derivative of a parent compound that has been prepared by theaddition of an acid. The pharmaceutically-acceptable salts include theconventional salts or the quaternary ammonium salts of the parentcompound formed, for example, from inorganic or organic acids. Forexample, such conventional salts include, but are not limited to, thosederived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfamic, phosphoric, nitric and the like; and the saltsprepared from organic acids such as acetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic,ethane disulfonic, oxalic, isethionic, and the like. Certain acidic orbasic compounds of the present invention may exist as zwitterions. Allforms of the compounds, including free acid, free base, and zwitterions,are contemplated to be within the scope of the present invention.

Description:

The present invention springs in part from the inventors' development ofcompstatin analogs displaying improvements in both inhibitory potencyand pharmacokinetic parameters. Selective modification of the compstatinN-terminus with non-proteinogenic amino acids and/or other molecularentities resulted in certain analogs with subnanomolar binding affinity(K_(D)=0.5 nM) and other similarly potent derivatives with improvedsolubility in clinically relevant solvents. Pharmacokinetic evaluationin non-human primates revealed plasma half-life values exceedingexpectations for peptidic drugs. Bioavailability evaluation in twonon-human primate models demonstrated subcutaneous, intramuscular andoral bioavailability of certain analogs.

One modification in accordance with the present invention comprisesadding a component to the N-terminus of compstatin(Ile-[Cys-Val-Val-Gln-Asp-Trp-Gly-His-His-Arg-Cys]-Thr; SEQ ID NO:1)that improves solubility and plasma stability of the peptide, whilemaintaining or improving C3 binding affinity and complement inhibitoryactivity. In particular embodiments, the added component is an aminoacid residue, particularly a residue that resist proteolytic cleavage,such as an N-methylated amino acid (e.g., N-methyl Gly (Sar), or aD-amino acid (e.g., D-Tyr). Also, as discussed in greater detail below,the D configuration of the N-terminal residue may better configure thefree amino group for polar interaction with C3. Additionally, aminoacids or analogs comprising a hydrophobic side chain (e.g., including anaromatic ring) at the N-terminus facilitates binding to C3, likely viainteraction with a hydrophobic pocket at the compstatin-C3 binding site.

Reference is made to the exemplary analogs set forth below, which showsignificantly improved activity over compstatin and even the potentanalog, Ac-Ile-[Cys-Val-Trp(Me)-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys]-Thr-NH₂(SEQ ID NO:2) (Katragadda et al., 2006, supra, WO 2007/062249; sometimesreferred to herein as “4 (1 MeW)”), as well as several other favorablecharacteristics discussed in detail below.

“Compstatin 30” (Cp30):Sar-Ile-[Cys-Val-Trp(Me)-Gln-Asp-Trp-Sar-Ala-His- Arg-Cys]-mIle-NH₂(SEQ ID NO: 7; also referred to in the Examples as “peptide 3”)“Compstatin 40” (Cp40):dTyr-Ile-[Cys-Val-Trp(Me)-Gln-Asp-Trp-Sar-Ala-His- Arg-Cys]-mIle-NH₂(SEQ ID NO: 18; also referred to in the Examples as “peptide 14”)

Without intending to be bound or limited by theory, it is believed thatthe improved C3 binding affinity of the analogs described herein is dueat least in part to higher affinity interactions mediated by theN-terminus. For instance, SPR and ELISA data indicate that D-aminoacids, or amino acids with hydrophobic side chains improved C3 binding,while the combination of features, i.e., D-amino acids with aromaticside chains e.g., D-Tyr), were most advantageous. In general, D-aminoacids with aromatic side chains were shown to be favored over aminoacids with shorter side chains in either D or L configuration.Furthermore, docking studies indicate that the improved affinity stemsfrom additional polar and non-polar interactions involving thepositioning of the free amino group, and the nature and positioning ofthe side chain on the N-terminal residue. For instance, the affinitygain of Cp40 (SEQ ID NO:18) was determined to be due at least in partfrom a combination of interactions with C3 at the N-terminus of theanalog; (1) the D configuration of the N-terminal Tyr better presentedthe free amino group for polar interaction with C3, a feature that alsoexplains the advantage of the D configuration overall; and (2) the bulkyhydrophobic side chain was able to fit into a hydrophobic pocket on C3cand also presented a hydroxyl group for hydrogen bonding with C3c. Inaddition, docking studies predicted an analog comprising Ac-Trp at theN-terminus (Example 2) to bind C3 with high affinity. SPR analysis ofpeptide 1 indeed showed high binding affinity, comparable to that ofCp40 (SEQ ID NO:18) (peptide 14). It was determined that both peptidesutilize the hydrophobic binding pocket on C3, C3b or C3c, proximal tothe N-terminus of compstatin.

N-methylation can affect a peptide in several ways. First, a potentialhydrogen bond donor is replaced with a methyl group, which cannot form ahydrogen bond. Second, the N-methyl group is weakly electron-donatingwhich means it can slightly increase the basicity of the neighboringcarbonyl group. Third, the size of the N-methyl group could cause stericconstraint, depending on the nature of the neighboring residues.Finally, the N-methylation can change the trans/cis population of theamide bond, thus changing local peptide conformation in a manner similarto a proline. In the case of Cp30 (SEQ ID NO:7), SPR data indicateslightly faster associate rate and slower dissociate rate than Cp20 (SEQID NO:3), which suggests that Cp30 (SEQ ID NO:7) has more favorable freesolution conformation for binding to C3/C3b/C3c and the binding isstronger. Considering the absence of an Ac group and the presence of amethyl group in the N-terminal position, it is reasonable to surmisethat the modification allowed the N-terminus to take part in strongerpolar interaction with residues S388/S437/D349 of C3c. This was madepossible by positioning the free N-terminus to a favorable position viaN-methylation in a way that enhances polar interactions with the bindingsite on C3/C3c.

In addition to improved C3 binding affinity, the analogs of the presentinvention possess improved solubility characteristics as compared withpreviously available analogs, such as Cp20 (SEQ ID NO:3). For systemicpharmacological administration, analogs with high solubility in bothwater for injection (WFI) and phosphate-buffered saline (PBS) aredesirable to minimize the required injection volume. By comparison,analogs with a high solubility in WFI and lower solubility in PBS couldproduce a more long-lasting gel, precipitate or suspension for topicalapplication or local injection, such as intraocular injection, e.g., fortreatment of AMD. It was determined that Cp30 (SEQ ID NO:7) was solublein both WFI and PBS, while Cp40 (SEQ ID NO:18) was less soluble in PBSthan in WFI.

The peptide analogs of the invention further display favorable plasmastability characteristics, believed to be due at least in part to thepresence of one or more N-terminal components that resist proteaseattack, e.g., a D-amino acid residue or an N-methyl group, oralbumin-binding molecules. In addition, the analogs bind specificallyand robustly to C3, C3b and C3c in plasma. Importantly, the stabilityafforded by the N-terminal and/or other modifications described hereincontribute to improved bioavailability from oral, subcutaneous orintramuscular administration, as demonstrated in mouse and two non-humanprimate model systems, as well as improved (i.e., slower) plasmaelimination half-live values of the analogs in vivo, as demonstrated inprimate model systems.

The above-described N-terminal modifications can be combined with othermodifications of compstatin previously shown to improve activity,thereby producing peptides with significantly improved complementinhibiting activity. For example, acetylation of the N-terminustypically increases the complement-inhibiting activity of compstatin andits analogs. Accordingly, addition of an acyl group at the aminoterminus of the peptide, including but not limited to N-acetylation, isone embodiment of the invention, though may not be needed if theN-terminus of the peptide is already stable, or if solubility becomes anissue.

As another example, it is known that substitution of Ala for His atposition 9 improves activity of compstatin and is a preferredmodification of the peptides of the present invention as well. It hasalso been determined that substitution of Tyr for Val at position 4 canresult in a modest improvement in activity (Klepeis et al., 2003, J AmChem Soc 125: 8422-8423).

It was disclosed in WO2004/026328 and WO2007/0622249 that Trp andcertain Trp analogs at position 4, as well as certain Trp analogs atposition 7, especially combined with Ala at position 9, yields many-foldgreater activity than that of compstatin. These modifications are usedto advantage in the present invention as well.

In particular, peptides comprising 5-fluoro-tryptophan or either5-methoxy-, 5-methyl- or 1-methyl-tryptophan, or 1-formyl-tryptophan atposition 4 have been shown to possess 31-264-fold greater activity thancompstatin. Particularly preferred are 1-methyl and 1-formyl tryptophan.It is believed that an indole ‘N’-mediated hydrogen bond is notnecessary at position 4 for the binding and activity of compstatin. Theabsence of this hydrogen bond or reduction of the polar character byreplacing hydrogen with lower alkyl, alkanoyl or indole nitrogen atposition 4 enhances the binding and activity of compstatin. Withoutintending to be limited to any particular theory or mechanism of action,it is believed that a hydrophobic interaction or effect at position 4strengthens the interaction of compstatin with C3. Accordingly,modifications of Trp at position 4 (e.g., altering the structure of theside chain according to methods well known in the art), or substitutionsat position 4 or position 7 of Trp analogs that maintain or enhance theaforementioned hydrophobic interaction are contemplated in the presentinvention as an advantageous modification in combination with themodifications at positions 8 and 13 as described above. Such analogs arewell known in the art and include, but are not limited to the analogsexemplified herein, as well as unsubstituted or alternativelysubstituted derivatives thereof. Examples of suitable analogs may befound by reference to the following publications, and many others:Beene, et al., 2002, Biochemistry 41: 10262-10269 (describing, interalia, singly- and multiply-halogenated Trp analogs); Babitzky &Yanofsky, 1995, J. Biol. Chem. 270: 12452-12456 (describing, inter alia,methylated and halogenated Trp and other Trp and indole analogs); andU.S. Pat. Nos. 6,214,790, 6,169,057, 5,776,970, 4,870,097, 4,576,750 and4,299,838. Trp analogs may be introduced into the compstatin peptide byin vitro or in vivo expression, or by peptide synthesis, as known in theart.

In certain embodiments, Trp at position 4 of compstatin is replaced withan analog comprising a 1-alkyl substituent, more particularly a loweralkyl (e.g., C₁-C₅) substituent as defined above. These include, but arenot limited to, N(α) methyl tryptophan and 5-methyltryptophan. In otherembodiments, Trp at position 4 of compstatin is replaced with an analogcomprising a 1-alkanoyl substituent, more particularly a lower alkanoyl(e.g., C₁-C₅) substituent as defined above, e.g., N(α) formyltryptophan, 1-acetyl-L-tryptophan and L-β-homotryptophan.

It was disclosed in WO2007/0622249 that incorporation of5-fluoro-tryptophan at position 7 in compstatin increased the enthalpyof the interaction between the resulting compstatin analog and C3,relative to compstatin, whereas incorporation of 5-fluoro-tryptophan atposition 4 in decreased the enthalpy of this interaction. Accordingly,modifications of Trp at position 7, as described in WO2007/0622249, arecontemplated as useful modifications in combination with the N-terminalmodifications described above.

Other modifications are described in WO2010/127336. One modificationdisclosed in that document comprises constraint of the peptide backboneat position 8 of the peptide. In a particular embodiment, the backboneis constrained by replacing glycine at position 8 (Gly⁸) with N-methylglycine. Another modification disclosed in that document comprisesreplacing Thr at position 13 with Ile, Leu, Nle (norleucine), N-methylThr or N-methyl Ile.

Still other modifications are described in co-pending Application No.61/385,711. One such modification comprises replacement of the C2-C12disulfide bond with addition of a CH₂ to form a homocysteine at C2 orC12, and introduction of a thioether bond, to form a cystathionine, suchas a gamma-cystathionine or a delta-cystathionine. Another modificationcomprises replacement of the C2-C12 disulfide bond with a thioether bondwithout the addition of a CH₂, thereby forming a lantithionine. Theanalogs comprising the thioether bond demonstrate activity that issubstantially the same as that of certain of the disulfide bond analogsand also possess equivalent or improved stability characteristics.

Yet other internal modifications are described in the presentapplication. For instance, substituting ornithine for arginine atposition 11, and/or substituting asparagine for aspartic acid atposition 6 of certain compstatin analogs (e.g. Cp20, SEQ ID NO:3, Cp40,SEQ ID NO:18), results in analogs with binding and complement inhibitoryactivity similar to the parent compounds. In addition, one or both ofthose substitutions is expected to render the analogs less susceptibleto metabolism by certain physiological enzymes found in the intestinaltract, liver or plasma.

The modified compstatin peptides of the present invention may beprepared by various synthetic methods of peptide synthesis viacondensation of one or more amino acid residues, in accordance withconventional peptide synthesis methods. For example, peptides aresynthesized according to standard solid-phase methodologies. Othermethods of synthesizing peptides or peptidomimetics, either by solidphase methodologies or in liquid phase, are well known to those skilledin the art. During the course of peptide synthesis, branched chain aminoand carboxyl groups may be protected/deprotected as needed, usingcommonly known protecting groups. An example of a suitable peptidesynthetic method is set forth in Example L Modification utilizingalternative protecting groups for peptides and peptide derivatives willbe apparent to those of skill in the art.

Alternatively, certain peptides of the invention may be produced byexpression in a suitable prokaryotic or eukaryotic system. For example,a DNA construct may be inserted into a plasmid vector adapted forexpression in a bacterial cell (such as E. coli) or a yeast cell (suchas Saccharomyces cerevisiae), or into a baculovirus vector forexpression in an insect cell or a viral vector for expression in amammalian cell. Such vectors comprise the regulatory elements necessaryfor expression of the DNA in the host cell, positioned in such a manneras to permit expression of the DNA in the host cell. Such regulatoryelements required for expression include promoter sequences,transcription initiation sequences and, optionally, enhancer sequences.

The peptides can also be produced by expression of a nucleic acidmolecule in vitro or in vivo. A DNA construct encoding a concatemer ofthe peptides, the upper limit of the concatemer being dependent on theexpression system utilized, may be introduced into an in vivo expressionsystem. After the concatemer is produced, cleavage between theC-terminal Asn and the following N-terminal G is accomplished byexposure of the polypeptide to hydrazine.

The peptides produced by gene expression in a recombinant procaryotic oreucaryotic system may be purified according to methods known in the art.A combination of gene expression and synthetic methods may also beutilized to produce compstatin analogs. For example, an analog can beproduced by gene expression and thereafter subjected to one or morepost-translational synthetic processes, e.g., to modify the N- orC-terminus or to cyclize the molecule.

Advantageously, peptides that incorporate unnatural amino acids, e.g.,methylated amino acids, may be produced by in vivo expression in asuitable prokaryotic or eukaryotic system. For example, methods such asthose described by Katragadda & Lambris (2006, Protein Expression andPurification 47: 289-295) to introduce unnatural Trp analogs intocompstatin via expression in E. coli auxotrophs may be utilized tointroduce N-methylated or other unnatural amino acids at selectedpositions of compstatin.

The structure of compstatin is known in the art, and the structures ofthe foregoing analogs are determined by similar means. Once a particulardesired conformation of a short peptide has been ascertained, methodsfor designing a peptide or peptidomimetic to fit that conformation arewell known in the art. Of particular relevance to the present invention,the design of peptide analogs may be further refined by considering thecontribution of various side chains of amino acid residues, as discussedabove (i.e., for the effect of functional groups or for stericconsiderations).

It will be appreciated by those of skill in the art that a peptide mimicmay serve equally well as a peptide for providing the specific backboneconformation and side chain functionalities required for binding to C3and inhibiting complement activation. Accordingly, it is contemplated asbeing within the scope of the present invention to produce C3-binding,complement-inhibiting compounds through the use of eithernaturally-occurring amino acids, amino acid derivatives, analogs ornon-amino acid molecules capable of being joined to form the appropriatebackbone conformation. A non-peptide analog, or an analog comprisingpeptide and non-peptide components, is sometimes referred to herein as a“peptidomimetic” or “isosteric mimetic,” to designate substitutions orderivations of the peptides of the invention, which possess the samebackbone conformational features and/or other functionalities, so as tobe sufficiently similar to the exemplified peptides to inhibitcomplement activation.

The use of peptidomimetics for the development of high-affinity peptideanalogs is well known in the art (see, e.g., Vagner et al., 2008, Curt.Opin. Chem. Biol. 12: 292-296; Robinson et al., 2008, Drug Disc. Today13: 944-951) Assuming rotational constraints similar to those of aminoacid residues within a peptide, analogs comprising non-amino acidmoieties may be analyzed, and their conformational motifs verified, byany variety of computational techniques that are well known in the art.

The modified compstatin peptides of the present invention can bemodified by the addition of polyethylene glycol (PEG) components to thepeptide. As is well known in the art, PEGylation can increase thehalf-life of therapeutic peptides and proteins in vivo. In oneembodiment, the PEG has an average molecular weight of about 1,000 toabout 50,000. In another embodiment, the PEG has an average molecularweight of about 1,000 to about 20,000. In another embodiment, the PEGhas an average molecular weight of about 1,000 to about 10,000. In anexemplary embodiment, the PEG has an average molecular weight of about5,000. The polyethylene glycol may be a branched or straight chain, andpreferably is a straight chain.

The compstatin analogs of the present invention can be covalently bondedto PEG via a linking group. Such methods are well known in the art.(Reviewed in Kozlowski A. et al. 2001, BioDrugs 15: 419-29; see also,Harris J M and Zalipsky S, eds. Polyethylene glycol), Chemistry andBiological Applications, ACS Symposium Series 680 (1997)). Non-limitingexamples of acceptable linking groups include an ester group, an amidegroup, an imide group, a carbamate group, a carboxyl group, a hydroxylgroup, a carbohydrate, a succinimide group (including withoutlimitation, succinimidyl succinate (SS), succinimidyl propionate (SPA),succinimidyl carboxymethylate (SCM), succinimidyl succinamide (SSA) andN-hydroxy succinimide (NHS)), an epoxide group, an oxycarbonylimidazolegroup (including without limitation, carbonyldimidazole (CDI)), a nitrophenyl group (including without limitation, nitrophenyl carbonate (NPC)or trichlorophenyl carbonate (TPC)), a trysylate group, an aldehydegroup, an isocyanate group, a vinylsulfone group, a tyrosine group, acysteine group, a histidine group or a primary amine. In certainembodiments, the linking group is a succinimide group. In oneembodiment, the linking group is NHS.

The compstatin analogs of the present invention can alternatively becoupled directly to PEG (i.e., without a linking group) through an aminogroup, a sulfhydryl group, a hydroxyl group or a carboxyl group. In oneembodiment, PEG is coupled to a lysine residue added to the C-terminusof compstatin.

As an alternative to PEGylation, the in vivo clearance of peptides canalso be reduced by linking the peptides to certain other molecules orpeptides. For instance, certain albumin binding peptides (ABP) displayan unusually long half-life of 2.3 h when injected by intravenous bolusinto rabbits (Dennis et al., 2002, J Biol Chem. 277: 35035-35043). Apeptide of this type, fused to the anti-tissue factor Fab of D3H44enabled the Fab to bind albumin while retaining the ability of the Fabto bind tissue factor (Nguyen et al., 2006, Protein Eng Des Set 19:291-297.). This interaction with albumin resulted in significantlyreduced in vivo clearance and extended half-life in mice and rabbits,when compared with the wild-type D3H44 Fab, comparable with those seenfor PEGylated Fab molecules, immunoadhesins, and albumin fusions.WO2010/127336 sets forth suitable synthesis strategies utilizing an ABPas well as an albumin-binding small molecule (ABM), and optionallyemploying a spacer or linker between the components. Those proceduresresulted in the production of conjugates of ABP- and ABM-compstatinanalogs capable of inhibiting complement activation and also exhibitingextended in vivo survival. Example 1 herein describes the use of thoseand other procedures with a higher affinity albumin-binding smallmolecule, ABM2, to generate a compstatin analog-ABM2 C-terminalconjugate utilizing a linker molecule. Example 1 further describes theproduction of N-terminal conjugates of certain compstatin analogs withthree different albumin-binding small molecules, ABM, ABM0 and ABM2using direct attachment without a linker. Such conjugates, whetherC-terminal, N-terminal direct or via a spacer or linker, display C3binding and complement-inhibiting activity comparable to or exceedingthat of the unconjugated analogs, as well as favorable in vivoretention.

The complement activation-inhibiting activity of compstatin analogs,peptidomimetics and conjugates may be tested by a variety of assaysknown in the art. In certain embodiments, the assays described in theExamples are utilized. A non-exhaustive list of other assays is setforth in U.S. Pat. No. 6,319,897, WO99/13899, WO2004/026328,WO2007/062249 and WO2010/127336, including, but not limited to, (1)peptide binding to C3 and C3 fragments; (2) various hemolytic assays;(3) measurement of C3 convertase-mediated cleavage of C3; and (4)measurement of Factor B cleavage by Factor D.

The peptides and peptidomimetics described herein are of practicalutility for any purpose for which compstatin itself is utilized, asknown in the art. Such uses include, but are not limited to: (1)inhibiting complement activation in the serum, and on cells, tissues ororgans of a patient (human or animal), which can facilitate treatment ofcertain diseases or conditions, including but not limited to,age-related macular degeneration, rheumatoid arthritis, spinal cordinjury, Parkinson's disease, Alzheimer's disease, cancer, sepsis,paroxysmal nocturnal hemoglobinuria, psoriasis and respiratory disorderssuch as asthma, chronic obstructive pulmonary disease (COPD), allergicinflammation, emphysema, bronchitis, bronchiecstasis, cystic fibrosis,tuberculosis, pneumonia, respiratory distress syndrome (RDS—neonatal andadult), rhinitis and sinusitis; (2) inhibiting complement activationthat occurs during cell or organ transplantation, or in the use ofartificial organs or implants (e.g., by time-restricted systemicadministration before, during and/or after the procedure or by coatingor otherwise treating the cells, organs, artificial organs or implantswith a peptide of the invention); (3) inhibiting complement activationthat occurs during extracorporeal shunting of physiological fluids(blood, urine) (e.g., by time-restricted systemic administration before,during and/or after the procedure or by coating the tubing through whichthe fluids are shunted with a peptide of the invention); and (4) inscreening of small molecule libraries to identify other inhibitors ofcompstatin activation (e.g., liquid- or solid-phase high-throughputassays designed to measure the ability of a test compound to competewith a compstatin analog for binding with C3 or a C3 fragment).

To implement one or more of the utilities mentioned above, anotheraspect of the invention features pharmaceutical compositions comprisingthe compstatin analogs or conjugates described and exemplified herein.Such a pharmaceutical composition may consist of the active ingredientalone, in a form suitable for administration to a subject, or thepharmaceutical composition may comprise the active ingredient and one ormore pharmaceutically acceptable carriers, one or more additionalingredients, or some combination of these. The active ingredient may bepresent in the pharmaceutical composition in the form of aphysiologically acceptable ester or salt, such as in combination with aphysiologically acceptable cation or anion, as is well known in the art.

A particular compstatin analog of the invention may be selected for aparticular formulation on the basis of its solubility characteristics.As mentioned above, analogs that are highly soluble in water or bufferedsaline may be particularly suitable for systemic injection because theinjection volume can be minimized. By comparison, analogs with highwater solubility and lower solubility in buffered saline could produce amore long-lasting gel, suspension or precipitate for topical applicationor local injection, such as intraocular injection. Thus, forillustrative purposes and not intended to be limiting, Cp30 (SEQ IDNO:7) could be selected for pharmaceutical formulations to beadministered by systemic injection, while Cp40 (SEQ ID NO:18) may beselected for formulations for intravitreal injection. Notably, Cp40 (SEQID NO:18) has been demonstrated to be available orally and viasubcutaneous or intramuscular injection, which provides importantadditional avenues for delivery, as discussed below.

The formulations of the pharmaceutical compositions may be prepared byany method known or hereafter developed in the art of pharmaceuticaltechnology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doesunit.

As used herein, the term “pharmaceutically-acceptable carrier” means achemical composition with which a compstatin analog may be combined andwhich, following the combination, can be used to administer thecompstatin analog to an individual.

As used herein, the term “physiologically acceptable” ester or saltmeans an ester or salt form of the active ingredient which is compatiblewith any other ingredients of the pharmaceutical composition, which isnot deleterious to the subject to which the composition is to beadministered.

The pharmaceutical compositions useful for practicing the invention maybe administered to deliver a dose of between 1 ng/kg and 100 mg/kg bodyweight as a single bolus, or in a repeated regimen, or a combinationthereof as readily determined by the skilled artisan. In certainembodiments, the dosage comprises at least 0.1 mg/kg, or at least 0.2mg/kg, or at least 0.3 mg/kg, or at least 0.4 mg/kg, or at least 0.5mg/kg, or at least 0.6 mg/kg, or at least 0.7 mg/kg, or at least 0.8mg/kg, or at least 0.9 mg/kg, or at least 1 mg/kg, or at least 2 mg/kg,or at least 3 mg/kg, or at least 4 mg/kg, or at least 5 mg/kg, or atleast 6 mg/kg, or at least 7 mg/kg, or at least 8 mg/kg, or at least 9mg/kg, or at least 10 mg/kg, or at least 15 mg/kg, or at least 20 mg/kg,or at least 25 mg/kg, or at least 30 mg/kg, or at least 35 mg/kg, or atleast 40 mg/kg, or at least 45 mg/kg, or at least 50 mg/kg, or at least55 mg/kg, or at least 60 mg/kg, or at least 65 mg/kg, or at least 70mg/kg, or at least 75 mg/kg, or at least 80 mg/kg, or at least 85 mg/kg,or at least 90 mg/kg, or at least 95 mg/kg, or at least 100 mg/kg, on adaily basis or on another suitable periodic regimen. In a particularembodiment, the dosage is between about 0.5 mg/kg and about 20 mg/kg, orbetween about 1 mg/kg and about 10 mg/kg, or between about 2 mg/kg andabout 6 mg/kg.

In one embodiment, the invention envisions administration of a dose thatresults in a serum concentration of the compstatin analog between about0.01 μM and about 30 μM in an individual. In certain embodiments, thecombined dose and regimen will result in a serum concentration, or anaverage serum concentration over time, of the compstatin analog of atleast about 0.01 μM, or at least about 0.02 or at least about 0.03 μM,or at least about 0.04 μM in or at least about 0.05 μM, or at leastabout 0.06 μM, or at least about 0.07 μM, or at least about 0.08 μM, orat least about 0.09 μM, or at least about 0.1 μM, 0.11 μM, or at leastabout 0.12 μM, or at least about 0.13 μM, or at least about 0.14 μM, orat least about 0.15 μM, or at least about 0.16 μM, or at least about0.17 μM, or at least about 0.18 μM, or at least about 0.19 μM, or atleast about 0.2 μM, or at least about 0.3 μM, or at least about 0.4 μM,or at least about 0.5 μM, or at least about 0.6 μM, or at least about0.7 μM, or at least about 0.8 μM, or at least about 0.9 μM, or at leastabout 1 μM or at least about 1.5 μM, or at least about 2 μM, or at leastabout 2.5 μM, or at least about 3 μM, or at least about 3.5 μM, or atleast about 4 μM, or at least about 4.5 μM, or at least about 5 μM, orat least about 5.5 μM, or at least about 6 μM, or at least about 6.5 μM,or at least about 7 μM, or at least about 7.5 μM, or at least about 8μM, or at least about 8.5 μM, or at least about 9 μM, or at least about9.5 μM, or at least about 10 μM, or at least about 10.5 μM, or at leastabout 11 μM or at least about 11.5 μM, or at least about 12 μM, or atleast about 12.5 μM, or at least about 13 μM, or at least about 13.5 μM,or at least about 14 μM, or at least about 14.5 μM, or at least about 15μM, or at least about 15.5 μM, or at least about 16 μM, or at leastabout 16.5 μM, or at least about 17 μM, or at least about 17.5 μM, or atleast about 18 μM, or at least about 18.5 μM, or at least about 19 μM,or at least about 19.5 μM, or at least about 20 μM, or at least about20.5 μM, or at least about 21 μM or at least about 21.5 μM, or at leastabout 22 μM, or at least about 22.5 μM, or at least about 23 μM, or atleast about 23.5 μM, or at least about 24 μM, or at least about 24.5 μM,or at least about 25 μM, or at least about 25.5 μM, or at least about 26μM, or at least about 26.5 μM, or at least about 27 μM, or at leastabout 27.5 μM, or at least about 28 μM, or at least about 28.5 μM, or atleast about 29 μM, or at least about 29.5 μM, or at least about 30 μM.In certain embodiments, the combined dose and regimen will result in aserum concentration, or an average serum concentration over time, of thecompstatin analog of up to about 0.1 μM, or up to about 0.11 μM, or upto about 0.12 μM, or up to about 0.13 μM, or up to about 0.14 μM, or upto about 0.15 μM, or up to about 0.16 μM, or up to about 0.17 μM, or upto about 0.18 μM, or up to about 0.19 μM, or up to about 0.2 μM, or upto about 0.3 μM, or up to about 0.4 μM, or up to about 0.5 μM, or up toabout 0.6 μM, or up to about 0.7 μM, or up to about 0.8 μM, or up toabout 0.9 μM, or up to about 1 μM or up to about 1.5 μM, or up to about2 μM, or up to about 2.5 μM, or up to about 3 μM, or up to about 3.5 μM,or up to about 4 μM, or up to about 4.5 μM, or up to about 5 μM, or upto about 5.5 μM, or up to about 6 μM, or up to about 6.5 μM, or up toabout 7 μM, or up to about 7.5 μM, or up to about 8 μM, or up to about8.5 μM, or up to about 9 μM, or up to about 9.5 μM, or up to about 10μM, or up to about 10.5 μM or up to about 11 μM or up to about 11.5 μM,or up to about 12 μM, or up to about 12.5 μM, or up to about 13 μM, orup to about 13.5 μM, or up to about 14 μM, or up to about 14.5 μM, or upto about 15 μM, or up to about 15.5 μM, or up to about 16 μM, or up toabout 16.5 μM, or up to about 17 μM, or up to about 17.5 μM, or up toabout 18 μM, or up to about 18.5 μM, or up to about 19 μM, or up toabout 19.5 μM, or up to about 20 μM, or up to about 20.5 μM or up toabout 21 μM or up to about 21.5 μM, or up to about 22 μM, or up to about22.5 μM, or up to about 23 μM, or up to about 23.5 μM, or up to about 24μM, or up to about 24.5 μM, or up to about 25 μM, or up to about 25.5μM, or up to about 26 μM, or up to about 26.5 μM, or up to about 27 μM,or up to about 27.5 μM, or up to about 28 μM, or up to about 28.5 μM, orup to about 29 μM, or up to about 29.5 μM, or up to about 20 μM.

Suitable ranges include about 0.1 to about 30 μM, or about 1 to about 29μM, or about 2 to about 28 μM, or about 3 to about 27 μM, or about 4 toabout 26 μM, or about 5 to about 25 μM, or about 6 to about 24 μM, orabout 7 to about 23 μM, or about 8 to about 22 μM, or about 9 to about21 μM, or about 10 to about 20 μM, or about 11 to about 19 μM, or about12 to about 18 μM, or about 13 to about 17 μM, or about 1 to about 5 μM,or about 5 to about 10 μM, or about 10 to about 15 μM, or about 15 toabout 20 μM, or about 20 to about 25 μM, or about 25 to about 30 μM.While the precise dosage administered will vary depending upon anynumber of factors, including but not limited to, the type of patient andtype of disease state being treated, the age of the patient and theroute of administration, such dosage is readily determinable by theperson of skill in the art.

The pharmaceutical composition can be administered to a patient asfrequently as several times daily, or it may be administered lessfrequently, such as once a day, once a week, once every two weeks, oncea month, or even less frequently, such as once every several months oreven once a year or less. The frequency of the dose will be readilyapparent to the skilled artisan and will depend upon any number offactors, such as, but not limited to, the type and severity of thedisease being treated, the type and age of the patient, as describedabove.

Pharmaceutical compositions that are useful in the methods of theinvention may be administered systemically in oral, parenteral,ophthalmic (including intravitreal), suppository, aerosol, topical,transdermal or other similar formulations. Such pharmaceuticalcompositions may contain pharmaceutically acceptable carriers and otheringredients known to enhance and facilitate drug administration. Otherformulations, such as nanoparticles, liposomes, resealed erythrocytes,and immunologically based systems may also be used to administer acompstatin analog according to the methods of the invention.

As used herein, “oral administration” or “enteral administration” of apharmaceutical composition includes any route of administrationcharacterized by introduction into the gastrointestinal tract. Suchadministration includes feeding by mouth as well as orogastric orintragastric gavage. Such administration also may include sublingual,buccal, intranasal, pulmonary or rectal administration, among otherroutes known in the art.

Formulations of a pharmaceutical composition suitable for oraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, in a variety of dosage forms,including but not limited to pills, tablets, granules, powders,capsules, dispersions, suspensions, solutions, emulsions,microemulsions, gels and films, to name a few. Such dosage formstypically include carriers and excipients to facilitate formulation anddelivery of the active ingredients.

The pharmaceutically acceptable carriers are selected from proteins,carbohydrates, lipids, organic and inorganic molecules, and combinationsthereof. The active ingredients can be combined with the carrier in anappropriate diluent to form a solution or a suspension. Such liquidformulations can be viscous or non-viscous depending on the amount andthe carrier used. The liquid formulations can be used directly or can befurther formulated into an appropriate capsule, gel capsule or solid bymethods know to those skilled in the art. Alternatively, solidformulations can be made by combining solid components. Such solidformulations can be used as a powder or formulated into granules,capsules, tablets or films any one of which can be made as a timerelease formulation.

Suitable proteins for use as carriers in oral dosage forms include milkproteins such as casein, sodium caseinate, whey, reduced lactose whey,whey protein concentrate, gelatin, soy protein (isolated), brown algaeprotein, red algae protein, baker's yeast extract and albumins. Suitablecarbohydrates include celluloses such as methylcellulose, sodiumcarboxymethylcellulose, carboxymethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, cellulose acetate and ethyl cellulose,starches such as cornstarch, potato starch, tapioca starch, wheatstarch, acid modified starch, pregelatinized starch and unmodifiedstarch, alginates such as ammonium alginate, sodium alginate, andcalcium alginate, glutens such as corn gluten and wheat gluten, gumssuch as acacia (gum Arabic), gum ghatti, guar gum, karaya gum (sterculiagum) and gum (tragacanth), insoluble glucose isomerase enzymepreparations, sugars such as corn sugar, invert sugar, corn syrup, highfructose corn syrup, and sodium gluconate. Suitable lipids includetocopherols such as a-tocopherol acetate, short-, medium- and long-chainfatty acids and esters thereof, fatty alcohols and ethers thereof, oilssuch as coconut oil (refined), soybean oil (hydrogenated) and rapeseedoil, aluminum palmitate, dilauryl thiodipropionate, enzyme-modifiedlecithin, calcium stearate, enzyme-modified fats, glycerylpalmitostereate, lecithin, mono- and diglycerides, glycerin and waxessuch as beeswax (yellow and white), candelilla wax and carnauba wax andvegetable oil. Suitable organic and inorganic substances include methyland vinyl pyrrolidones such as polyvinylpyrrolidone, methylsulfonylmethane, dimethylsulfoxide and related compounds, hydroxy andpolyhydroxy acids such as polylactic acid, among many others.

In some embodiments, controlled release forms may be prepared to achievea sustained, or location-specific liberation of the compstatin analog inthe digestive tract in order to improve absorption and prevent certainforms of metabolism. For example, acid-resistant coatings of tablet oracid-resistant capsule materials may be used to prevent a release ofcompstatin analogs in the stomach and protect the compound frommetabolism by gastric enzymes. Suitable materials and coatings toachieve controlled release after passage of the stomach are primarilycomposed of fatty acids, waxes, shellac, plastics and plant fibers andinclude, but are not limited to, methyl acrylate-methacrylic acidcopolymers, cellulose acetate succinate, hydroxy propyl methyl cellulosephthalate, hydroxy propyl methyl cellulose acetate succinate(hypromellose acetate succinate), polyvinyl acetate phthalate, sodiumalginate or stearic acid. Sustained release in the gastrointestinaltract can for example be achieved by embedding compstatin analogs in amatrix of insoluble substances such as various acrylics, chitin andothers. Methods to prepare such formulations are known to those skilledin the art.

Compstatin may be formulated into suppositories or clysters for rectal,vaginal or urethral administration. For this purpose, compstatin analogscan be dissolved or suspended in a greasy base carrier such as cocoabutter that is solid or semi-solid at room temperature but melts at bodytemperature or in a water-soluble solid base such as polyethylene glycolor glycerin (made from glycerol and gelatin). Other excipients may beadded to improve the formulation, and suppositories will be shaped in aform that facilitates administration. In other embodiments, liquidsuppositories consisting of compstatin analogs dissolved or suspended ina liquid carrier suitable for rectal delivery to be applied with a smallsyringe may be used.

For the treatment of chronic or acute lung conditions in whichcomplement activation is implicated, a preferred route of administrationof a pharmaceutical composition is pulmonary administration.Accordingly, a pharmaceutical composition of the invention may beprepared, packaged, or sold in a formulation suitable for pulmonaryadministration via the buccal cavity. Such a formulation may comprisedry particles which comprise the active ingredient and which have adiameter in the range from about 0.5 to about 7 nanometers, andpreferably from about 1 to about 6 nanometers. Such compositions areconveniently in the form of dry powders for administration using adevice comprising a dry powder reservoir to which a stream of propellantmay be directed to disperse the powder or using a self-propellingsolvent/powder-dispensing container such as a device comprising theactive ingredient dissolved or suspended in a low-boiling propellant ina sealed container. Preferably, such powders comprise particles whereinat least 98% of the particles by weight have a diameter greater than 0.5nanometers and at least 95% of the particles by number have a diameterless than 7 nanometers. More preferably, at least 95% of the particlesby weight have a diameter greater than 1 nanometer and at least 90% ofthe particles by number have a diameter less than 6 nanometers. Drypowder compositions preferably include a solid fine powder diluent suchas sugar and are conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic or solid anionic surfactant or a solid diluent(preferably having a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may also provide the active ingredient in the form of dropletsof a solution or suspension. Such formulations may be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, including replacement pulmonary surfactant, or a preservativesuch as methylhydroxybenzoate. The droplets provided by this route ofadministration preferably have an average diameter in the range fromabout 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary deliveryare also useful for intranasal delivery of a pharmaceutical compositionof the invention.

Another formulation suitable for intranasal administration is a coarsepowder comprising the active ingredient and having an average particlefrom about 0.2 to 500 micrometers. Such a formulation is administered inthe manner in which snuff is taken i.e. by rapid inhalation through thenasal passage from a container of the powder held close to the nares.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient, and may further comprise one or more of theadditional ingredients described herein.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, intravenous,subcutaneous, intraperitoneal, intramuscular, intraarticular,intravitreal, intrasternal injection, and kidney dialytic infusiontechniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e. powder or granular) form for reconstitution with asuitable vehicle (e.g. sterile pyrogen-free water) prior to parenteraladministration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution can be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, in microbubbles forultrasound-released delivery or as a component of a biodegradablepolymer systems. Compositions for sustained release or implantation maycomprise pharmaceutically acceptable polymeric or hydrophobic materialssuch as an emulsion, an ion exchange resin, a sparingly soluble polymer,or a sparingly soluble salt.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agentsincluding replacement pulmonary surfactants; dispersing agents; inertdiluents; granulating and disintegrating agents; binding agents;lubricating agents; sweetening agents; flavoring agents; coloringagents; preservatives; physiologically degradable compositions such asgelatin; aqueous vehicles and solvents; oily vehicles and solvents;suspending agents; dispersing or wetting agents; emulsifying agents,demulcents; buffers; salts; thickening agents; fillers; emulsifyingagents; antioxidants; antibiotics; antifungal agents; stabilizingagents; and pharmaceutically acceptable polymeric or hydrophobicmaterials. Other “additional ingredients” which may be included in thepharmaceutical compositions of the invention are known in the art anddescribed, for example in Genaro, ed., 1985, Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa.

Methods:

Another aspect of the invention features methods of regulatingcomplement activation. In general, the methods comprise contacting amedium in which regulation of complement activation is desired with acompstatin analog of the present invention, wherein the contactingresults in regulation of complement activation in the medium. The mediumcan be any medium in which regulation of complement activation isdesired. In certain embodiments, the medium includes cells or tissues ofan organism, including (1) cultured cells or tissues, (2) cells ortissues within the body of a subject or patient, and (3) cells ortissues that have been removed from the body of one subject and will bereplaced into the body of the same patient (e.g., extracorporealshunting of blood or autologous transplantation) or transferred toanother patient. In connection with the latter embodiment, the mediummay further comprise a biomaterial, such as tubing, filters or membranesthat contact the cells or tissues during extracorporeal shunting.Alternatively, the medium may comprise biomaterials that are implantedinto a subject.

In certain embodiments, the methods of regulating complement activationapply to living patients or subjects and comprise part or all of amethod of treating the patient for a pathological condition associatedwith complement activation, particularly AP-mediated complementactivation. Many such pathological conditions are known in the art (see,e.g., Holers, 2008, supra) and include, but are not limited to, asatypical hemolytic uremic syndrome (aHUS), dense deposit disease,age-related macular degeneration (AMD), paroxysomal nocturnalhemoglobinuria (PNH), cold agglutinin disease (CAD) rheumatoid arthritis(RA), systemic lupus erythematosus (SLE), several autoimmune andautoinflammatory kidney diseases, autoimmune myocarditis, multiplesclerosis, traumatic brain and spinal cord injury, intestinal and renalischemia-reperfusion (IR) injury, spontaneous and recurrent pregnancyloss, anti-phospholipid syndrome (APS), Alzheimer's disease, asthma,anti-nuclear cytoplasmic antigen-associated pauci-immune vasculitis(Wegener's syndrome), non-lupus autoimmune skin diseases such aspemphigus, bullous pemphigoid, and epidermolysis bullosa, post-traumaticshock, certain forms of cancer, and atherosclerosis. In particularembodiments, the pathological condition has been associated withmutations and polymorphisms in the gene encoding FH and/or CD46,including but not limited to: AMD, aHUS and membrano-proliferativeglomerulonephritis type II (MPGN-II, also referred to as dense depositdisease (DDD)). In other embodiments, the comstatin analogs of thepresent invention are suitable for use as a substitute for Eculizumab orTT30 in treatment of diseases for which those agents are currentlyprescribed, or for which they are being developed in pre-clinical andclinical studies. Those diseases include, but are not limited to, aHUS,PNH, CAD and AMD.

The treatment methods typically comprise (1) identifying a subject witha disease or condition treatable by regulation of complement activationas described hereinabove, and (2) administering to the subject aneffective amount of a compstatin analog of the invention using atreatment regimen and duration appropriate for the condition beingtreated. Development of appropriate dosages and treatment regimens willvary depending upon any number of factors, including but not limited to,the type of patient and type of disease state being treated, the age ofthe patient and the route of administration. The skilled artisan isfamiliar with the design of dosage regimens that take such variablesinto account. For instance, it will be apparent to the skilled artisanthat oral administration of a compstatin analog of the invention willrequire a higher initial dosage, due to the lesser bioavailability fromthat route as compared with, e.g., intravenous injection.

The following examples are provided to describe the invention in greaterdetail. They are intended to illustrate, not to limit, the invention.

Example 1

This example describes the synthesis of compstatin analogs withN-terminal modifications, and conjugates of certain analogs toalbumin-binding small molecules.

Chemicals.

Rink amide MBHA resin, Oxyma and the following Fmoc-amino acids wereobtained from Novabiochem (San Diego, Calif.): Ile, Cys(Trt), Val,Tyr(tBu), Gln(Trt), Asp(OtBu), Trp(Boc), Gly, Sar, Ala, MeAla, His(Trt),Arg(Pbf), MeIle, Phe, MePhe and D-Cha. DIC and Fmoc-Trp(Me)-OH werepurchased from AnaSpec (San Jose, Calif.). HOAt was purchased fromAdvanced ChemTech (Louisville, Ky.). NMP and DCM were obtained fromFisher Scientific (Pittsburgh, Pa.). All other chemical reagents forsynthesis were purchased from Sigma-Aldrich (St. Louis, Mo.) and usedwithout further purification.

Peptide Synthesis and Purification.

All peptides were synthesized manually by Fmoc solid-phase methodologyusing DIC and Oxyma as coupling reagents. The following procedures wereused for the synthesis of the linear peptides: Rink amide MBHA resin(0.59 mmol/g) was placed into a peptide synthesis glass vessel equippedwith frits on the bottom and swollen in DCM for 30 min. After removal ofthe Fmoc protecting group (25% piperidine in NMP, 5 and 10 min), theresin was washed 7 times with NMP and twice with DCM, and the individualamino acids were coupled to the resin. For each coupling, 3 equivalentsof the amino acid, HOAt, and DIC were used, with 10 min preactivation inNMP. All couplings were performed for 1 h and monitored by either theKaiser test or the chloranil test. In case of a positive test result,the coupling was repeated until a negative test result was observed. Thesynthesis was stopped after the coupling of Cys in position 1. Then theresin was split in HSW polypropylene syringe with frits on the bottom(Torviq, Niles, Mich.) and coupling of addition amino acids was coupledusing method reported previously.

Upon completion of the solid phase synthesis, the resin was washed fourtimes with NMP, DCM, and DCM/diethylether (1:1), and dried under highvacuum for 4 h. The peptides were cleaved from the resin with a mixtureof 94% TFA, 2.5% water, and 2.5% EDT and 1% TIPS for 2 h. Afterevaporation of the TFA under vacuum, the peptides were precipitated andwashed three times with cold diethyl ether. The liquid was separatedfrom the solid by centrifugation and decanted. The crude peptides weredried in vacuum and dissolved in 30% acetonitrile. The pH of solutionwas adjusted to 8-9 using concentrated ammonium hydroxide. To thesolution was added diluted hydrogen peroxide (1:100, 2 eq.) withvigorous stirring. The cyclization was monitored by using MALDI-TOF.Once the reaction was completed, the solution was supplemented with TFAto lower the pH to 2. And the solution was lyophilized. The crudepeptide was purified with RP-HPLC as described previously (Qu et al.,2011, supra). The purified peptides were >95% pure as determined byanalytical RP-HPLC (Phenomenex 00G-4041-E0 Luna 5μ C18 100A column,250×4.60 mm; Phenomenex, Torrance, Calif.). The mass of each peptide wasconfirmed using Waters MALDI micro MX instruments or Synapt HDMS.

Certain of the compstatin analogs were conjugated to an albumin-bindingsmall molecule, examples of which are shown below.

In one construct, ABM2 was coupled to the C-terminus of peptide Cp30(SEQ ID NO:7; Table 1 below) via a mini-PEG-3 spacer in accordance withthe methods described in WO2010/127336.

In other constructs, ABM, ABM0 or ABM2 were coupled to the N-terminus ofCP20 (SEQ ID NO:3) or CP40 (SEQ ID NO:18) without a spacer.

Example 2

Compstatin analogs synthesized by the methods described in Example 1were measured for C3 binding and complement-inhibitory activity.

Materials and Methods:

Inhibition of Complement Activation.

The ability of the compstatin analogs to inhibit the activation of theclassical pathway of complement was assessed by ELISA as describedelsewhere (Katragadda et al., 2006, supra; Mallik et al., 2005, supra).The percent inhibition was plotted against the peptide concentration,and the resulting data set was fitted to the logistic dose-responsefunction using Origin 8.0 software. IC₅₀ values were obtained from thefitted parameters that produced the lowest χ2 value. Each analog wasassayed at least three times.

SPR Analysis.

The interaction of the compstatin analogs with C3b was characterizedusing a Biacore 3000 instrument (GE Healthcare, Corp., Piscataway,N.J.). The running buffer was PBS, pH 7.4 (10 mM sodium phosphate, 150mM NaCl) with 0.005% Tween-20. Biotinylated C3b was capturedsite-specifically on a streptavidin chip at about 3000 and 5000 RUdensity; two untreated flow cells were used as reference surface. Forkinetic analysis, sets of five increasing concentrations of a particularcompound were injected over the chip surface one after the other in asingle cycle. Three-fold dilution series (0.49-40 nM were injected at 3μl/min; each injection was done for 2 min, allowing every time thepeptide to dissociate for 5 min before the next injection started. Afterthe end of the last injection, 40 min of dissociation time was allowed.Peptide 4 (1 MeW) was included in each experimental series as aninternal control and reference. Data analysis was performed usingScrubber (BioLogic Software, Campbell, Australia) and BiaEvaluation (GEHealthcare, Corp., Piscataway, N.J.). The signals from an untreated flowcell and an ensemble of buffer blank injections were subtracted tocorrect for buffer effects and injection artifacts. Processed biosensordata were globally fitted to a 1:1 Langmuir binding model (kindlyprovided by GE Healthcare), and the equilibrium dissociation constant(K_(D)) was calculated from the equation K_(D)=k_(d)/k_(a). Each assaywas performed at least twice.

Docking Peptides to C3c.

AutoDock Vina (Trott and Olson, 2010) was used for docking studies. Withexception of the backbone of the cyclic core region that can only behandled as rigid by Vina, all other parts of the peptides (terminalresidues, side chains) were defined as flexible during the docking runs.The residues of C3c near the N-terminus of analog Cp20 (SEQ ID NO:3)(i.e., Asp349, Lys386, Ser388, Asn390, Ser437, Asn452, Leu454, Asp491and Leu492) were defined as flexible for the docking experiments inorder to allow for more reasonable interactions between the extendedN-terminus of these peptides and C3c. The only exception was peptide 19,the N-terminus of which does not extended as other peptides; the bindingsite area on C3c therefore remained rigid in the docking of peptide 19.Initial structures of all peptides were manually built in PyMol based onthe C3c-bound structure of 4W9A. AutoDockTools was used to define thebinding pocket and prepare the initial structures of C3c and allpeptides from the pdb format into the input format of Vina (pdbqt).55 Inthe comparison plot of computational versus experimental binding freeenergy (ΔG), the experimental ΔG was calculated from the affinity valuesdetermined by SPR as ΔG=RT ln(KD), with R=1.986 cal K⁻¹ mol⁻¹ andT=293.15 K.

Results:

Structure/Activity of N-Terminal Extensions.

Using a molecular modeling approach, the early compstatin analog 4W9Awas replaced by Cp20 (SEQ ID NO:3) in the co-crystal structure with thetarget fragment C3c. Computational analysis of this complex confirmedthat the methyl group of Sar8 forms a contact with oxygen atom of G489in C3c (distance ˜4.0 Å). Yet analysis of the binding site also revealedthe existence of a hydrophobic area on C3c that may be exploited viaN-terminal extension of the peptide ligand. While not buried in thebinding pocket of C3c, the N-terminus of compstatin has previously beenprotected by an acetyl moiety primarily to improve peptide stability;however, such capping also had a beneficial effect on the inhibitorypotency. Based on the current lead compound Cp20 (SEQ ID NO:3), theeffect of replacing the N-terminal acetyl moiety on target binding wasevaluated (Table 1). For this purpose, analogs were subjected toquantitative kinetic profiling for their binding to C3b and compared tothe clinically used analog 4 (1 MeW) and to Cp20 (SEQ ID NO:3) (Table 1,FIG. 1). Indeed, substitution of the terminal acetyl with a shortermethyl group (peptide 1) led to a drop in affinity by almost an order ofmagnitude, below that of 4 (1 MeW), thereby confirming the advantage ofN-terminal capping. In contrast, capping with a glycine residue (peptide2) improved the dissociation rate (k_(d)) yet slightly lowered theassociation rate (k_(a)), leading to only a very small net change inaffinity (compared to Cp20 (SEQ ID NO:3)). N-methylation of Gly to Sar(peptide 3) restored the association properties while retaining thebeneficial dissociation value, which produced a compound withsignificantly improved affinity (K_(D)=1.6 nM; Table 1).

To further explore the benefit of N-terminal optimization, additionalCp20 (SEQ ID NO:3)-based analogs with natural (peptides 4-8), methylated(peptides 9-13) and D-amino acids (peptides 14-18) at position Xaa0(FIG. 1B; Table 1) were screened. The set included representativehydrophobic, hydrophilic, and charged side chains. All tested compoundsshowed strong binding (K_(D)<20 nM), with the k_(a) values(1-4×10⁶M⁻¹s⁻¹) showing less variability than k_(d) values (1-25×10⁻³s⁻¹) across the entire panel (Table 1, FIG. 1B). All analogs followed a1:1 Langmuir kinetic model when screened for binding to C3b, therebystrongly supporting the presence of a single high-affinity binding site.In general, D-amino acids with hydrophobic side chains appeared to befavored over the acetyl (Ac) moiety of Cp20 (SEQ ID NO:3). Among those,peptide 14 with a DTyr at that position was the most potent, with asubnanomolar affinity (K_(D)=0.5 nM; Table 1) and the slowestdissociation rate of the panel. The affinity of peptides in which Ac wasreplaced by other amino acids fell between that of peptides 1 and 14,with most analogs clustering around the profile of Cp20 (SEQ ID NO:3)(FIG. 1B). Tyrosine appears generally preferred since all peptides withN-terminal Tyr, its O-methyl analog and its D-isoform ranked among thebest binders with affinities around or below 1 nM. In contrast, residueswith shorter side chains like Gly, Thr, or Ala derivatives seemed lessfavorable and did not improve the affinity compared to Cp20 (SEQ IDNO:3). Thus, replacement of the capping Xaa0 residue appears to be welltolerated for a wide range of amino acid residues with varyingproperties, from hydrophobic to charged.

TABLE 1 Evaluation of kinetic parameters and inhibitory potency for aseries of compstatin analogs(Xaa0-Xaa1-[Cys-Val-Trp(Me)-Gln-Asp-Trp-Sar-Ala-His-Arg-Cys]-mIle-NH₂)(SEQ ID NO: 4) with modifications at the N-terminus. k_(a): associationrate; k_(d): dissociation rate; K_(D): binding constant from SPR; IC₅₀,peptide concentration to reach 50% inhibition of classical pathwaycomplement activation. ND: not determined. SEQ Peptide ID NO: XaaO Xaa1k_(a) (10⁶/Ms) k_(d) (10⁻³/s) K_(D) (nM) IC₅₀ (nM) 4(1MeW)^(a) 2 — — 1.1± 0.1 11.3 ± 0.9  10.3 ± 1.5  132 ± 7  Cp20^(b) 3 Ac Ile 1.9 4.0 2.4  15 Me Ile 1.3 ± 0.3 24.8 ± 7.3  18.6 ± 3.5  180 ± 17  2 6 Gly Ile 1.2 ±0.3 2.9 ± 0.2 2.5 ± 0.5 113 ± 16  3^(c) 7 Sar Ile 1.9 ± 0.5 2.9 ± 0.31.6 ± 0.3  82 ± 14  4 8 Tyr Ile 2.1 ± 0.3 2.5 ± 0.1 1.2 ± 0.1  72 ± 10 5 9 Phe Ile 2.1 ± 0.4 3.3 ± 0.3 1.6 ± 0.2 ND  6 10 Arg Ile 1.7 ± 0.22.9 ± 0.2 1.7 ± 0.2 ND  7 11 Trp Ile 1.6 ± 0.1 3.6 ± 0.2 2.2 ± 0.2 ND  812 Thr Ile 1.2 ± 0.1 3.1 ± 0.2 2.6 ± 0.3 ND  9 13 Tyr(Me) Ile 2.3 ± 0.42.6 ± 0.1 1.2 ± 0.2 ND 10 14 mPhe Ile 1.6 ± 0.2 2.9 ± 0.3 1.8 ± 0.3 ND11 15 mVal Ile 1.8 ± 0.3 3.5 ± 0.6 1.9 ± 0.1 ND 12 16 mIle Ile 1.6 ± 0.23.7 ± 0.3 2.4 ± 0.5 ND 13 17 mAla Ile 1.4 ± 0.2 3.4 ± 0.3 2.5 ± 0.6 ND14^(c) 18 D-Tyr Ile 2.8 ± 0.5 1.4 ± 0.1 0.5 ± 0.1 66 ± 8 15 19 D-Phe Ile2.3 ± 0.3 2.6 ± 0.0 1.1 ± 0.1 ND 16 20 D-Trp Ile 2.0 ± 0.2 2.4 ± 0.1 1.2± 0.1 ND 17 21 D-Cha² Ile 1.8 ± 0.5 2.7 ± 0.4 1.5 ± 0.2 ND 18 22 D-AlaIle 1.4 ± 0.3 3.4 ± 0.4 2.5 ± 0.4 ND 19 23 Ac Trp 3.8 ± 0.3 1.7 ± 0.30.5 ± 0.1 ND 20 24 Tyr Gly 2.1 ± 0.3 7.3 ± 1.4 3.5 ± 0.3 ND^(a)Ac-Ile-[Cys-Val-Trp(Me)-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys]-Thr-NH₂(Katragadda et al., 2006, supra, WO 2007/062249; sometimes referred toherein as “4(1MeW)”); included as a standard in all analyses but doesnot follow the Cp20 (SEQ ID NO: 3) template. ^(b)Base compound forN-terminal modifications; binding/potency values from previouspublication (WO2010/127336). ^(c)Selected for further testing: peptide3 - Cp30 (SEQ ID NO: 7); peptide 14 - Cp40 (SEQ ID NO: 18)

Computational Analysis.

Extended docking analyses were performed to provide structural evidencefor the observed effects on binding affinity and generate acomputational model for predicting novel analogs. Initially, the dockingstrategy was validated using the data set from the screening ofN-terminally modified analogs of Cp20 (SEQ ID NO:3) (peptides 1-18;Table 1). For this purpose, the compounds were prepared in silico,docked into the compstatin binding pocket of human C3c (Janssen et al.,2007, supra), and the binding free energy (ΔG) was calculated andcompared to the SPR affinity-derived values by determining the Pearson'scoefficient (R, FIG. 2). The overall correlation between experimentaland calculated ΔG values was 0.46 based on five independent dockingstudies over the entire data set (FIG. 2). Out of the 19 analogs in thedata sets, three peptides bearing either a very short moiety (methyl;peptide 1) or aromatic natural amino acid (peptides 5 and 7) showed asignificantly higher deviation; when these analogs were excluded, thecorrelation increased to 0.69 (FIG. 2).

A more detailed analysis of the docked peptides indicated that most ofthe N-terminally modified compstatin analogs formed additional contactswith a polar area and a shallow pocket on C3c. For example, the polararea involving Asp349, Ser388 and Ser437 of C3c interacts with theN-terminal amino group of DTyr in peptide 14 (FIG. 3A). In contrast,such a polar interaction is not favored for peptides carrying naturalamino acid residues at this position, as exemplified for peptide 4, dueto a different orientation of the amino group (FIG. 2A). Furthermore,the side chain of the elongated amino acid (DTyr) in peptide 14 formsadditional hydrophobic contacts with Leu454 and Leu492 in the shallowextended pocket on C3c. Finally, the hydroxyl group of DTyr formed aweak hydrogen bond with Asn452 of C3c. A combination of those effects islikely to contribute to the observed subnanomolar binding affinity ofpeptide 14.

To further explore distinct strategies of addressing the N-terminalpocket, two analogs were designed in which an aromatic residue waslocated at position Xaa0 or Xaa1 (peptides 19 and 20; Table 1). Based onthe computational model developed above, the side chain of the new Trpin peptide 19 was predicted to fit well into the hydrophobic bindingpocket (FIG. 3B), whereas a short flexible Gly linker was chosen inpeptide 20 to allow a better orientation of the Tyr side chain whencompared to the homolog peptide 4. While peptide 20 showed a threefoldweaker binding affinity than peptide 4, peptide 19 reached sub-nanomolarbinding affinities (K_(D)=0.5 nM; Table 1), making it as potent aspeptide 14. Together, these results demonstrate the advantage of aproperly oriented hydrophobic residue adjacent to Cys at position 2.

Additional analogs were constructed based on Cp40 (peptide 14, SEQ IDNO:18, Table 1). These are shown in Table 2 below.

TABLE 2 Evaluation of kinetic parameters and inhibitory potency foranalogs based on Cp40 (SEQ ID NO: 18) with modifications within thepeptide. Numbering within the peptide designation indicates the positionrelative to compstatin. k_(a): association rate; k_(d): dissociationrate; K_(D): binding constant from SPR; IC₅₀, peptide concentration toreach 50% inhibition of classical pathway complement activation. SEQk_(a) k_(d) K_(D) IC₅₀ Peptide ID NO: (10⁶/Ms) (10⁻³/s) (nM) (nM) Cp4018 2.8 ± 0.6 1.3 ± 0.2 0.5 ± 0.1 0.14 ± 0.05 (peptide 14) Cp40 25 2.5 ±0.1 2.0 ± 0.1 0.8 ± 0.1 0.22 (11Orn) Cp40 26 0.9 ± 0.1 2.8 ± 0.1 3.0 ±0.4 0.26 (6Asn) Cp40 27 2.0 ± 0.4 2.8 ± 0.6 1.5 ± 0.4 0.36 (11Orn 6Asn)^(a) ornithine substituted for arginine at position 11. ^(b) asparaginesubstituted for aspartic acid at position 6.

As mentioned above, ABM, ABM0 or ABM2 were coupled without a spacer tothe N-terminus of CP20 (SEQ ID NO:3) or CP40 (SEQ ID NO:18) and certainvariants thereof. Those analogs displayed binding and complementinhibitory activity in the same range as the Cp40 analog and itsderivatives set forth in Table 2.

Example 3

Certain of the compstatin analogs synthesized as described in Example 1were measured for solubility in water for injection (WFI) and Dulbecco'sPBS (DPBS).

Materials and Methods:

Approximately 5 mg of each peptide (acetate form) was weighed out intoseparate LoBind Eppendorf tubes and 50 μL water for injection (WFI) wasadded to each tube. Each sample was centrifuged at 13000 rpm for 2 minand diluted for measuring the optical density (OD) at 280 nm using aNanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, Del.).Each concentrated sample was diluted 1:20 into Dulbecco's phosphatebuffered saline (DPBS, without potassium and calcium; Invitrogen,Carlsbad, Calif.). The samples were monitored for precipitation, andeach sample was vortexed for 5 min and centrifuged at 13000 rpm for 2min. The OD of each DPBS supernatant was measured to determine peptideconcentration at saturation.

Results:

While the presence of three acidic or basic residues (Asp6, His10,Arg11) in most compstatin analogs contributes to a generally favorablesolubility in aqueous solutions, their zwitterionic nature maynegatively affect solubility in buffered solutions. Accordingly, thesolubility of selected compounds in two clinically relevant solvents,i.e., water for injection (WFI) and Dulbecco's PBS (DPBS) was evaluated.In addition, the ultra performance liquid chromatography (UPLC)retention time of these peptides on a C18 column was measured to reflecttheir apparent relative hydrophobicity (Table 3).

TABLE 3 Solubility of peptides in WFI (Water for Injection) and DPBS,and UPLC (Ultra Performance Liquid Chromatography) retention time as anindication of hydrophobicity. SEQ Solubility (mg/mL)^(a)Hydophobicity^(b) Peptide ID NO: WFI DPBS pH 7.4 Retention Time (min)4(1MeW) 2 >50 3.5 5.09 Cp20 3 13 2.7 5.33 Cp30 7 >50 6.9 4.60 Cp4018 >50 0.8 4.73 Peptide 19^(c) 23 ND <0.2 ND ^(a)Measured as OD (280 nm)at saturation; WFI = water for injection, DPBS = Dubelcco's phosphatebuffered saline ^(b)Measured as retention time during UPLC analysis on aC18 column ^(c)Peptide 19 could not be solubilized at 100 μM or above inPBS during ELISA studies.

The solubility in WFI was excellent, with values exceeding 50 mg/mL forall compounds with the exception of Cp20 (SEQ ID NO:3). In general, thesolubility in DPBS was significantly lower for all analogs. Thedecreased solubility of Cp20 (SEQ ID NO:3) in both solvents, as comparedto 4 (1 MeW), is considered a consequence of its hydrophobicity arisingfrom two N-methylations (positions 8 and 13) and the C-terminalThr-to-Ile substitution. The replacement of the N-terminal acetyl moietyin 4 (1 MeW) and Cp20 (SEQ ID NO:3) by uncapped amino acid residuesinduced a significant gain in hydrophilicity for Cp30 (SEQ ID NO:7) andCp40 (SEQ ID NO:18) and restored their high solubility in WFI (>50mg/mL). However, the incorporation of a hydrophobic DTyr at itsN-terminus negatively impacted the solubility of Cp40 (SEQ ID NO:18)(0.8 mg/mL) in DPBS. In contrast, the presence of a small N-terminal Sarin Cp30 (SEQ ID NO:7) largely improved its solubility in DPBS (6.9mg/mL), rendering this peptide almost twice as soluble as theclinically-used 4 (1 MeW) analog.

Example 4

Certain of the compstatin analogs synthesized as described in Example 1were measured for plasma stability and plasma protein binding in humanplasma.

Materials and Methods:

Plasma Stability.

Fresh human plasma containing lepirudin (3.75 units/ml) was incubated at37° C. with Cp30 (SEQ ID NO:7), Cp40 (SEQ ID NO:18) or control peptide2B at a final concentration of 20 μM each. Samples of 100 μL were takenfor solid phase extraction. A 96-well plate HLB Oasis 30 μm 10 mg(Waters, Milford, Mass.) was employed for extraction. The SPE materialwas conditioned by addition of 500 μL each of methanol and ACN followedby addition of 500 μL of milli-Q water. Sample was diluted 1:1 with 4%H₃PO₄. After loading the sample, washing was carried out twice with 500μL of 10% ACN in 0.1% formic acid. Sample was eluted with 200 μL of 65%ACN in 0.1% formic acid and collected in the Eppendorf LoBind collectionplate. Sample for UPLC-MS was diluted 1:10 in milli-Q water with 0.1%formic acid. Cp20 (SEQ ID NO:3) was spiked in each sample before SPE asan internal standard.

Plasma Protein Binding.

Cp30 (SEQ ID NO:7) was spiked in 500 μL of fresh human plasma containinglepirudin (3.75 units/ml) so that the final peptide concentration was 20μM (C3: 1.2 mg/mL, 6.4 μM). A control sample was prepared in the sameway using Cp30 (SEQ ID NO:7) and milli-Q water to determine the area ofpeptide in UPLC-MS at 1 μM. The plasma sample was equilibrated at roomtemperature for 10 min. Then, 500 μL of 30% PEG in milli-Q water(MW3350) was slowly added to the plasma sample while mixing. The mixturewas centrifuged at 14000 rpm for 10 min to separate the supernatant. Thepellet was dissolved in 1000 μL of FPLC buffer A and separated by FPLCusing Mono Q 5/5 column and fractions was collected at 1 mL per tube 0.5mL of each fraction was mixed with same volume off 4% H₃PO₄ for SPE andUPLC-MS analysis.

UPLC-MS analysis. UPLC-MS analysis was performed on a SYNAPT HDMS(Waters, Milford, Mass.) equipped with an ESI source controlled byMassLynx 4.1 software (Waters). Each sample was injected inquadruplicates. An online ACQUITY UPLC (Waters) system was used forpeptide separation by reversed-phase liquid chromatography. Thecapillary voltage was 3.2 kV, the cone voltage was 30 V and the sourcetemperature was 120° C. [Glu1]-fibrinogen peptide was used for lock-masscorrection with a sampling rate of 30 s. Mass spectra were acquired inpositive mode over an m/z range 200-2000 Da at scan rate 1 s. Thepresence of the analyte was confirmed by retention time and mass.Selectivity was studied by analysis of blank plasma sample and purepeptides to determine the presence of any interference coeluting withthe analyte. After injection, analytes were separated on a 1.7 μm UPLCBEH130 C18 column (Water, 2.1 μm×150 mm, part number 186003556). Theanalytical column temperature was held at 40° C. Peptides were separatedat flow rate 0.3 mL/min. The gradient was linear 10-60% B (0.1% formicacid in acetonitrile) over 8 min.

Results:

Plasma Stability.

To investigate the stability of the new analogs with free N-terminus,Cp30 (SEQ ID NO:7) and Cp40 (SEQ ID NO:18) were selected for incubationin human plasma at 37° C. (FIG. 4A). The control linear peptide 2B(LRFLNPFSLDGSGFW, SEQ ID NO:28) was cleaved quickly upon contact withplasma. The zero time point sample showed cleavage at the Arg position.The peptide completely disappeared within 30 min. Under the sameconditions, both Cp30 (SEQ ID NO:7) and Cp40 (SEQ ID NO:18) showedremarkable stability in plasma. More than 55% of peptides remain after 5days. The UPLC-MS chromatograms at time 0, 24 and 120 h are quitesimilar (FIG. 4B). No major cleavage product was observed.

Plasma Protein Binding.

To investigate the binding specificity of Cp30 (SEQ ID NO:7), excessivepeptide was incubated in fresh human plasma. Plasma proteins wereprecipitated with PEG3350 and separated using a small Mono Q column.Each 1 mL fraction was measured for the presence of Cp30 (SEQ ID NO:7).Fractions that contained Cp30 (SEQ ID NO:7) were further analyzedquantitatively using UPLC-MS and tested for the identity of theco-eluting protein. It was found that 7.5% of the Cp30 (SEQ ID NO:7) waslocated in the flow-through while 88.0% and 4.5% co-eluted with C3 andC3c, respectively. The identity of the proteins was identified bySDS-PAGE followed by Coomassie staining and Western Blot. In addition,the total amount of Cp30 (SEQ ID NO:7) detected was equal to the amountof plasma C3.

Example 5

Compstatin analogs Cp20 (SEQ ID NO:3), Cp30 (SEQ ID NO:7) and Cp40 (SEQID NO:18), synthesized as described in Example 1, were measured for invivo retention in a cynomolgus monkey model. The binding profiles of thepeptides were compared in plasma of four primate species: human,cynomolgus monkey, rhesus monkey and baboon, using the SPR methoddescribed above.

Materials and Methods:

Primate Studies and Sample Collection.

Evaluation of plasma half-life and generation of major metabolites wasperformed at the Simian Conservation Breeding and Research Center(SICONBREC, Makati City, Philippines) in cynomolgus monkeys (Macacafascicularis). For each analog (Cp20 (SEQ ID NO:3), Cp30 (SEQ ID NO:7)and Cp40 (SEQ ID NO:18)), two healthy animals were sedated and injectedintravenously with 2 mg/kg of the compound (dissolved in saline forinjection). Blood samples (1-2 mL) were collected immediately before andat various time points after compound injection (2, 5 and 30 min; 1, 2,4, 6, and 24 hours) in EDTA-coated Vacutainer tubes to preventcoagulation and complement activation, and centrifuged at ˜800×g for 10min to obtain plasma. Plasma samples were immediately frozen and storedfor further analysis. All NHP studies were performed in accordance withanimal welfare laws and regulations.

Analysis of Plasma Samples.

Prior to analysis by UPLC-MS, compstatin analogs in the plasma sampleswere extracted by solid phase extraction (SPE) in a 96-well plate format(HLB Oasis 30 μm, 10 mg; Waters, Milford, Mass.). The SPE material wasthoroughly conditioned using acetonitrile and water. Plasma samples werediluted 1:1 with 4% phosphoric acid, and a constant concentration ofCp20 (SEQ ID NO:3) (5 μM) was spiked into all samples containing Cp30(SEQ ID NO:7) or Cp40 (SEQ ID NO:18) as an internal standard; in thecase of Cp20 (SEQ ID NO:3)-containing samples, Cp40 (SEQ ID NO:18) wasused as internal standard. The samples were loaded on the SPE plate andwashed with 10% acetonitrile in 0.1% formic acid. Extracted peptideswere eluted with 200 μL of 65% acetonitrile in 0.1% formic acid andcollected in a LoBind tube (Eppendorf) to avoid peptide adsorption.Finally, 5 μL of each eluent was diluted with 45 μL 0.1% formic acid andinjected into the UPLC-MS system consisting of an online ACQUITY UPLCcoupled to a SYNAPT G2-S HDMS instrument equipped with an ESI source andcontrolled by MassLynx 4.1 software (Waters). Each sample was injectedin quadruplicates. Reversed-phase liquid chromatography was used forpeptide separation with a 1.7 μM UPLC BEH130 C18 column (2.1 μm×150 mm;Waters) at a column temperature of 40° C. Peptides were separated at aflow rate of 0.15 mL/min with a linear gradient of 10-60% acetonitrilein water containing 0.1% formic acid over 8 min. Eluted peptides weredirectly analyzed by HDMS; the ESI source capillary voltage was set to3.2 kV, the cone voltage to 30 V and the source temperature to 120° C.[Glu1]-fibrinopeptide B (Sigma) was used for lock-mass correction with asampling rate of 30 s. Mass spectra were acquired in positive mode overan m/z range of 50-1950 Da at a scan rate of 1 s.

Determination of Plasma Half-Life.

Calibration curves were prepared on the day of the analysis by spikingcompstatin analogs (Cp20 (SEQ ID NO:3), Cp30 (SEQ ID NO:7) and Cp40 (SEQID NO:18)) into freshly-thawed plasma from untreated cynomolgus monkeysat final concentrations of 0.5, 1, 2, 4, and 8 μM. All calibrationsamples were subjected to SPE and measured using UPLC-HDMS as describedabove. MS peak areas were determined by integration and plotted againstthe concentration, resulting in calibration curves that showed goodlinearity with regression coefficients (R2) greater than 0.993. For thepharmacokinetic analysis, the plasma concentration (Cp) at each timepoint was calculated from the extracted peak area of each peptide usingthe corresponding standard curve. The elimination constant (k_(e)) andplasma half-life (t_(1/2)) were determined from the slope of theterminal elimination phase (0.5-24 h) using the following equations:ln(Cp)=ln(Cp0)−k_(e)×t, and t₁₂=0.693/k_(e) Determination of C3 levels.The plasma concentration of C3 in individual cynomolgus monkeys used inthis study was determined by ELISA. Briefly, 96-well plates (MaxiSorp;Nunc) were coated with 1 μg/ml of a monoclonal anti-C3 antibody (clone8E11; Tosic et al., 1989, J Immunol. Methods 120: 241-249) in PBSovernight at 4.25° C. Wells were washed with PBS/Tween 0.05% and blockedwith PBS/BSA 1% for 1 h at room temperature. Plasma (diluted 1:10,000and 1:20,000 in PBS/BSA) or serial dilutions of purified cynomolgusmonkey C3 were then incubated for 1 h at room temperature followed bywashing and incubation with peroxidase-conjugated anti-C3 (MPBiomedicals, Solon, Ohio) diluted 1:1,000 in PBS/BSA for 1 h at roomtemperature. The reaction was developed using tetramethylbenzidinesubstrate (R&D Systems, Minneapolis, Minn.) per manufacturer'sinstructions and optical density was determined using a microplatereader with wavelength set at 450 nm.

Hemolytic Assay.

Rabbit erythrocytes were washed with phosphate-buffered saline (PBS)followed by washing with Veronal-buffered saline (VBS)^(Mg+)/EGTA. A1:20 dilution was prepared in VBS buffer. Plasma samples (1:10 inVBS—100 μl) was incubated with the rabbit erythrocytes solution (50 μl)in a 96-well plate at 37° C. for 1 h. EDTA (0.2 mM-15 μl) was added tostop the reaction and plate was centrifuged (2500×g 3 min) Supernatant(100 μl) was transferred to a new well and optical density was measuredat 405 nm. Incubation of erythrocytes with water or buffer was used aspositive (100% lysis) and negative (0% lysis) control, respectively.

Binding Profiles.

For the NHP specificity experiments, C3 from human, cynomolgus monkey,rhesus monkey, and baboon plasma was immobilized on individual flowcells of CM5 sensor chips (GE Healthcare) using standard amine couplingto reach target densities of 6,000-7,000 RU. Peptides Cp20 (SEQ ID NO:3)and Cp40 (SEQ ID NO:18) were quantitatively evaluated using a singlecycle kinetic approach as described in Example 2. To visually comparethe kinetic profiles independently of differences in target density oractivity, each binding curve was normalized to the maximum response andsuperimposed in Origin.

Results:

Peptidic drugs are often hampered by comparatively fast elimination fromplasma, which may be highly restrictive in clinical applications thatrely on constant systemic drug levels (e.g., PNH in the case ofcomplement inhibitors). A comparative study including Cp20 (SEQ ID NO:3)and the newly developed Cp30 (SEQ ID NO:7) and Cp40 (SEQ ID NO:18) wasperformed, in which cynomolgus monkeys were intravenously injected with2 mg/kg of each analog and the plasma levels were assessed by LC-MS overa period of 24 hours. All tested analogs followed a similar biphasicelimination profile, in which the plasma levels dropped more rapidlywithin the first hour after injection and then followed a much slowerdecrease throughout the later time points (FIG. 5A). The peptideconcentrations at which the kinetic change occurred were very similar tothat of the expected physiological plasma levels of the target proteinC3. Indeed, measurement of the C3 levels in the involved monkeys byELISA (4.9-12.8 μM) confirmed that the initial drop in compstatin levelsslowed down within the determined range of C3 (FIG. 5A). Theseobservations suggest a target-driven elimination model, where tightbinding to the abundant target C3 largely influences peptide excretion.Indeed, when the plasma half-life was calculated based on the terminallog-linear portion (1-24 h), a direct correlation to the bindingaffinity for C3 could be observed with half-life values of 9.3, 10.1 and11.8 h for Cp20 (SEQ ID NO:3), Cp30 (SEQ ID NO:7) and Cp40 (SEQ IDNO:18), respectively (FIG. 5B). The half-life of the Cp30-ABM2 conjugatewas observed to be 22 hours (not shown).

Concentrations of compstatin analogs were measured against inhibition ofcomplement activation via the alternative pathway in the plasma samplesusing an erythrocyte hemolytic assay. Complement inhibitory activity wasobserved to closely track the concentration of analog in the samples ateach time point measured.

Given the strong apparent dependence of the major elimination phase withthe binding affinity, the translation of these NHP-based studies to thehuman system appear to be influenced by the differential affinity ofthese compstatin analogs for human and NHP C3. Hence, the bindingprofiles of the peptides for C3 from humans and three relevant NHPs(cynomolgus monkey, rhesus monkey, baboon) was measured, using the SPRmethod described above. Both the affinity and kinetic profiles for allanalogs were highly comparable (FIG. 5C).

Example 6

Compstatin analog Cp40 (SEQ ID NO:18), synthesized as described inExample 1, was measured for bioavailability from subcutaneous and oralroutes of administration in a cynomolgus monkey model.

Materials and Methods:

Primate Studies and Sample Collection.

Evaluation of bioavailability was performed at the Simian ConservationBreeding and Research Center (SICONBREC, Makati City, Philippines) incynomolgus monkeys (Macaca fascicularis). Two healthy animals were usedfor each route of administration. The animals were sedated and injectedsubcutaneously with 2 mg/kg of the compound or orally by intragastricgavage with 4 mg/kg of the compound. Blood samples (1-2 mL) werecollected immediately before and at various time points after compoundinjection (2, 5 and 30 min; 1, 2, 4, 6, and 24 hours) in EDTA-coatedVacutainer tubes to prevent coagulation and complement activation, andcentrifuged at ˜800×g for 10 min to obtain plasma. Plasma samples wereimmediately frozen and stored for further analysis. All NHP studies wereperformed in accordance with animal welfare laws and regulations.

Analyses.

Analysis of plasma samples, determination of plasma half-life andcomplement inhibitory activity in plasma were performed as described inExample 5.

Results:

Peptidic drugs typically are very poorly bioavailable by any routeexcept intravenous administration, which is expensive, not welltolerated by patients, and usually needs to be performed by a trainedspecialist. The compstatin analog Cp40 (SEQ ID NO:18) was tested forbioavailability following subcutaneous or oral delivery. Cynomolgusmonkeys were subcutaneously injected with 2 mg/kg or orally injectedwith 4 mg/kg of the analog and the plasma levels were assessed by LC-MSover a period of 24 h. Results are shown in FIG. 6.

The plasma concentration of Cp40 (SEQ ID NO:18) reached its peak ofapproximately 12.5 μM within 4-5 hours following administration bysubcutaneous injection (FIG. 6, top panel). Oral injection of the analogresulted in a plasma concentration of approximately 0.023 μM within onehour of injection (FIG. 6, bottom panel; note the oral injection wassuccessful on only one of the two monkeys). By comparison (FIG. 5B),intravenous injection of that analog resulted in peak plasmaconcentrations of approximately 28 μM immediately following injection.

Concentrations of Cp40 (SEQ ID NO:18) were measured against inhibitionof complement activation via the alternative pathway in the plasmasamples from the subcutaneous injection using an erythrocyte hemolyticassay. Complement inhibitory activity was observed to closely track theconcentration of analog in the samples at each time point measured.

Example 7

Compstatin analog Cp30 (SEQ ID NO:7) and the Cp-30-ABM2 conjugatedescribed in Example 1, were measured for in vivo retention in a baboonmodel.

Materials and Methods:

Juvenile baboons (P. Anubis, Baboon Research Resources, University ofOklahoma) weighing 5-8 kg were used. Two baboons were used for thestudy, one for each compound. Each animal received a bolus dose ofpeptide (10 mg) by injection through the peripheral vein. Blood samplesfor the LC-MS/MS assay were collected in 1-ml plastic tubes containing50 μg lepirudin, and centrifuged at 2000 g for 20 min at 4° C. forplasma separation. Plasma samples were stored at −70° C. Blood sampleswere collected at pre-determined time intervals after injection of Cp30(SEQ ID NO:7) or the Cp30 (SEQ ID NO:7)-ABM2 conjugate. Samples weretreated with SPE and analyzed using LC-MS/MS. Calibration curves werecreated using standard peptides at various concentrations in plasma todetermine peptide concentration in each sample.

Extraction of compstatin analogs from plasma samples by SPE. A 96-wellplate HLB Oasis 30 μm 10 mg (Waters, Milford, Mass.) was employed forextraction. The SPE material was conditioned by addition of 500 μl ofmethanol, ACN followed by addition of 500 μL of milli-Q water. Sampleswere diluted with 4% H₃PO₄. After loading a sample, washing was carriedout with 500 μL of water and 10% ACN with 0.1% formic acid. Samples wereeluted with 200 μL of 65% ACN in 0.1% formic acid and collected in thecollection plate. Samples for LC-MS were diluted 1:2 to 1:11 in milli-Qwater with 10% ACN with 0.1% formic acid. CP20 (SEQ ID NO:3) was spikedin each sample before SPE, as an internal standard.

LC-MS/MS Analysis.

LC-MS/MS analysis was performed on a SYNAPT HDMS (Waters, Milford,Mass.) equipped with an ESI source controlled by MassLynx 4.1 software(Waters). Each sample was injected in triplicate. An online ACQUITY UPLC(Waters) system was used for peptide separation by reversed-phase liquidchromatography. The capillary voltage was 3.2 kV, the cone voltage was30 V and the source temperature was 120° C. [Glu1]-fibrinogen peptidewas used for lock-mass correction with a sampling rate of 30 s. Massspectra were acquired in positive mode over an m/z range 500-1800 Da atscan rate of is. The presence of the analyte was confirmed by retentiontime and mass. After injection, analytes were separated on a 1.7 μm UPLCBEH130 C18 column (Water, 1.0 μm×100 mm). The analytical columntemperature was held at 40° C. Peptides were separated at flow rate 0.15mL/min. The gradient was linear 15-55% B (0.1% formic acid inacetonitrile) over 7 min.

Results:

The plasma concentrations of peptide Cp30 (SEQ ID NO:7) and the ABM2conjugate were determined using LC-MS/MS after an intravenous bolusinjection into baboons. Peptide Cp30 (SEQ ID NO:7) displayed a half-lifeof 5 hours and Cp30 (SEQ ID NO:7)-ABM2 displayed a half-life of 7.5 hr.By comparison, in the same baboon model, compstatin analog 4 (1 MeW) anda potent analog (peptide 3) disclosed in WO2010/127336 were previouslydetermined to have half-lives of approximately 60-90 minutes.

Example 8

Compstatin analog Cp40 (SEQ ID NO:18), synthesized as described inExample 1, was measured for bioavailability from an intramuscular routeadministration in a baboon model.

Methods:

A juvenile baboon was injected intramuscularly with 2 mg/kg Cp40 (SEQ IDNO:18) Blood samples for the LC-MS/MS assay were collected in 1-mlplastic tubes containing 50 μg lepirudin, and centrifuged at 2000 g for20 min at 4° C. for plasma separation. Plasma samples were stored at−70° C. Blood samples were collected at pre-determined time intervalsafter injection of the analog. Samples were treated with SPE andanalyzed using LC-MS/MS. Calibration curves were created using standardpeptides at various concentrations in plasma to determine peptideconcentration in each sample.

Extraction of compstatin analog from plasma samples and LC-MS/MSanalysis were performed as described in Example 7. A hemolytic assay wasperformed as described in Example 5.

Results:

Results are shown in FIG. 7. The plasma concentration of Cp40 (SEQ IDNO:18) reached its peak of approximately 10 μM within about 5-6 hoursfollowing administration by intramuscular injection. Complementinhibitory activity was observed to closely track the concentration ofanalog in the samples at each time point measured.

The present invention is not limited to the embodiments described andexemplified above, but is capable of variation and modification withinthe scope of the appended claims.

1. A compound comprising a modified compstatin peptide (ICVVQDWGHHRCT(cyclic C2-C12; SEQ ID NO:1) or analog thereof, wherein the modificationcomprises an added or substituted N-terminal component that improves (1)the peptide's C3, C3b or C3c binding affinity, (2) the peptide'ssolubility in aqueous liquids, and/or (3) the peptide's plasma stabilityand/or plasma residence time, as compared with an unmodified compstatinpeptide under equivalent conditions.
 2. The compound of claim 1, whereinthe added component is an amino acid other than L-Gly, or non-peptideanalog of an amino acid.
 3. The compound of claim 2, wherein the addedcomponent is a D-amino acid.
 4. The compound of claim 2, wherein theadded component comprises at least one aromatic ring.
 5. The compound ofclaim 2, wherein the added component is D-Tyr.
 6. The compound of claim2, wherein the amino acid is N-methylated.
 7. The compound of claim 6,wherein the amino acid is N-methyl glycine (Sar).
 8. The compound ofclaim 2, wherein the added component is D-Tyr, D-Phe, Tyr(Me), D-Trp,Tyr, D-Cha, Cha, Phe, Sar, Arg, mPhe, mVal, Trp, mIle, D-Ala, mAla, Thror Tyr.
 9. The compound of claim 1, comprising a substituted N-terminalcomponent wherein Ile at position 1 is replaced with Ac-Trp or adipeptide Tyr-Gly.
 10. The compound of claim 1, further comprising oneor more modifications selected from: (a) replacement of His at position9 with Ala; (b) replacement of Val at position 4 with Trp or an analogof Trp; (c) replacement of Trp at position 7 with an analog of Trp; (d)modification of Gly at position 8 to constrain the backbone conformationat that location; (e) replacement of the Thr at position 13 with Ile,Leu, Nle, N-methyl Thr or N-methyl Ile; (f) replacement of the disulfidebond between C2 and C12 with a thioether bond to form a cystathionine ora lantithionine; (g) replacement of the Arg at position 11 with Orn; and(h) replacement of the Asp at position 6 with Asn.
 11. (canceled) 12.The compound of claim 10, wherein: (a) the analog of Trp at position 4is 1-methyl Trp or 1-formyl Trp; (b) the analog of Trp at position 7 isa halogenated Trp; and (c) the backbone is constrained by replacing theGly at position 8 (Gly8) with N^(α)-methyl Gly. 13-20. (canceled) 21.The compound of claim 1, which is a compstatin analog comprising apeptide having a sequence of SEQ ID NO:29, which is:Xaa1-Xaa2-Cys-Val-Xaa3-Gln-Xaa4-Xaa5-Gly-Xaa6-His-Xaa7-Cys-Xaa8, inwhich Gly between Xaa4 and Xaa5 optionally is modified to constrain thebackbone conformation; wherein: Xaa1 is absent or is Tyr, D-Tyr or Sar;Xaa2 is Ile, Gly or Ac-Trp; Xaa3 is Trp or an analog of Trp, wherein theanalog of Trp has increased hydrophobic character as compared with Trp;Xaa4 is Asp or Asn Xaa5 is Trp or an analog of Trp comprising a chemicalmodification to its indole ring wherein the chemical modificationincreases the hydrogen bond potential of the indole ring; Xaa6 is His,Ala, Phe or Trp; Xaa7 is Arg or Orn; and Xaa8 is Thr, Ile, Leu, Nle,N-methyl Thr or N-methyl Ile, wherein a carboxy terminal —OH of any ofthe Thr, Ile, Leu, Nle, N-methyl Thr or N-methyl Ile optionally isreplaced by —NH₂, and the peptide is cyclic via a Cys-Cys or thioetherbond.
 22. The compound of claim 21, wherein: the Gly at position 8 isN-methylated; Xaa1 is D-Tyr or Sar; Xaa2 is Ile; Xaa3 is Trp,1-methyl-Trp or 1-formyl-Trp; Xaa5 is Trp; Xaa6 is Ala; and Xaa8 is Thr,Ile, Leu, Nle, N-methyl Thr or N-methyl Ile with optional replacement ofthe carboxy terminal —OH with —NH₂.
 23. The compound of claim 22,wherein Xaa8 is Ile, N-methyl Thr or N-methyl Ile with optionalreplacement of the carboxy terminal —OH with —NH₂.
 24. The compound ofclaim 23, which comprises SEQ ID NO:7 or SEQ ID NO:18.
 25. The compoundof claim 1, further comprising an additional component that increasesthe bioavailability or extends the in vivo retention of the compound.26. The compound of claim 25, wherein the additional component isselected from: (a) polyethylene glycol (PEG) (b) an albumin bindingsmall molecule; and (c) an albumin binding peptide.
 27. (canceled) 28.The compound of claim 26, wherein the albumin binding small molecule isattached to the peptide at the N-terminus or at the C-terminus.
 29. Thecompound of claim 26, comprising a spacer between the peptide and thealbumin binding small molecule.
 30. (canceled)
 31. A pharmaceuticalcomposition comprising a and a pharmaceutically acceptable carrier,wherein the compound comprises a modified compstatin peptide(ICVVQDWGHHRCT (cyclic C2-C12; SEQ ID NO:1) or analog thereof, whereinthe modification comprises an added or substituted N-terminal componentthat improves (1) the peptide's C3, C3b or C3c binding affinity, (2) thepeptide's solubility in aqueous liquids, and/or (3) the peptide's plasmastability and/or plasma residence time, as compared with an unmodifiedcompstatin peptide under equivalent conditions.
 32. The pharmaceuticalcomposition of claim 31, formulated for administration of the compoundby a route selected from oral, topical, pulmonary, subcutaneous,intramuscular and intravenous. 33-38. (canceled)